Automated pelletized sample vision inspection apparatus and methods

ABSTRACT

A method for visually inspecting pelletized samples. The method generates an inspection image of the bottom of a sample tube holder. The sample tube holder has a number of sample tube locations, and each sample tube location is configured to receive a sample tube in it. The inspection image is evaluated to determine whether a tube image exists for each sample tube location, and each sample tube image is evaluated to determine whether a pellet is located in each sample tube.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to automated systems and methods forpreparing samples, such as biological samples. Particular exemplaryembodiments relate to processing samples used to determine the presenceor absence of human papillomavirus or other conditions.

2. Description of the Related Art

A wide variety of processing protocols are used in many different fieldsof art. Processing protocols are created and followed to help make suresimilar items are processed the same way. The use of protocols helpsprovide consistent processing results and, where the results are notconsistent, ensures that the differences are not attributable tovariances caused by the processing itself.

Processing protocols are particularly important in the field ofanalytical biological science, in which biological samples are takenfrom a subject and processed to diagnose medical conditions, such as thepresence or absence of a pathogen or viral infection. In many cases, itcan be difficult, burdensome, uncomfortable, or even painful to obtain asample from the subject, and therefore a high value is placed on takinggreat care with handling and testing the sample to prevent the need formultiple sample collection procedures. It is also may be desirable toperform as many tests as possible on the sample, and therefore thesample may need to be processed into multiple different sample aliquotsto be tested using multiple different protocols. As a result, it isdesirable to process as little of the sample as possible, to permitretests and alternative tests of a single collected sample. The desireto use smaller portions of each sample can place even stricterboundaries or requirements on sample processing protocols.

In some cases, analytical protocols may be regulated by governmententities. For example, some testing protocols must be approved by theUnited States Food and Drug Administration before they can be introducedinto commercial use. In such cases, the protocol must be followed notonly as a matter of sound scientific principles, but also to stay withinthe scope of government-regulated activities.

One exemplary sample processing protocol is the QIAGEN Hybrid Capture® 2(“HC2”) nucleic acid hybridization assay. This protocol is usedprimarily for detecting human papillomavirus (“HPV”) infections. The HC2assay is an in vitro assay, in which RNA probes are hybridized withtarget DNA, the RNA:DNA hybrids are captured onto a solid phase, and thecaptured RNA:DNA hybrids are detected with multiple antibodiesconjugated to alkaline phosphatase (a.k.a., signal amplification). Theparticular chemical and biological details of this process are known inthe art and need not be detailed herein. The HC2 assay may be performedmanually or through a combination of manual and automated processes. Themanual sample preparation protocol for the HC2 assay includes a seriesof manual steps, which include (in general terms): reagent preparation,sample mixing/aliquoting, pelleting/decanting, denaturing, and transfer.The process begins with a sample collected from a subject and containedin a vial of preservative fluid (e.g., PreservCyt® or SurePath™). Thedetails of the manual HC2 protocol steps, as performed on PreservCyt®samples, follow.

The reagent preparation step begins by adding 5 drops of indicator ordye to a denaturation reagent (“DNR”) causing the DNR to turn darkpurple. Next, the specimen transport medium (“STM”) and DNR are combinedin a 2:1 ratio and mixed by vortexing.

The sample mixing/aliquoting step is performed by vigorously shaking thePreservCyt® solution vial by hand or using a vortex mixer at maximumspeed setting. Immediately after mixing, a volume of the PreservCyt®specimen solution is pipetted and delivered to the bottom of a conicalsample processing container. The container is polypropylene, and may bea 10 milliliter Sarstedt conical tube or a 15 milliliter VWR or Corningbrand conical tube.

The pelleting/decanting step involves a number of substeps. First, apredetermined amount of sample conversion buffer (e.g., 0.4 millilitersadded to 4.0 milliliters of specimen for 1-2 tests per sample forsamples in PreservCyt® media) is added to the processing tube, and thenthe tube is capped and thoroughly mixed using a vortex mixer with a cupattachment. Next, the tube is centrifuged in a swinging bucket rotor at2,900 (±150)×g for 15 (±2) minutes. Following centrifuging, the operatorvisually verifies that a pink/orange cell pellet is present in thebottom of the tube. Even if no pellet is detected, the protocolcontinues, a pellet that is too small to see can still provide apositive test result (however, if there is no visible pellet, a negativetest result might be dismissed as a false negative, and such anindeterminate result may require further testing). Next, the supernatantis carefully decanted by inverting the tube and gently blotting(approximately 6 times) on absorbent low-lint paper towels until liquidno longer drips from the tube. Each blot is done on a clean area of thetowel. During blotting, the operator observes the tube to ensure thatthe cell pellet does not slide down the tube.

The denaturing step also includes a number of substeps. The step beginsby adding a volume of the STM/DNR mixture (prepared in the reagentpreparation step) to the pellet (e.g., 150 microliters of a 2:1 mixtureof STM and DNR per 4 milliliter sample). Next, the pellets areresuspended by vortexing the tube. The operator may individually vortexthe tube, or vortex it with other tubes on a MST Vortexer 2 machine. Ineither case, the tube is vortexed for at least 30 seconds at the highestspeed setting. If the pellet is difficult to resuspend, it may bevortexed an additional 10-30 seconds or until the pellet floats loosefrom the bottom of the tube. After vortexing, the tube is placed in arack, and the rack is placed in a 65° (±2°) Celsius water bath (withsufficient water to cover the liquid in the tube) for 15 (±2) minutes.Next, the tube is removed from the water bath, the exterior is dried,and the tube is vortexed again for 15-30 seconds (or, if it is vortexedon a MST Vortexer 2, for 1 minute) at the highest speed setting. Afterthe second vortexing, the tube is again placed in a rack that is placedin a 65° (±2°) Celsius water bath (with sufficient water to cover theliquid in the tube) for 30 (±3) minutes. Following the second water bathtubes that were vortexed on a MST Vortexer 2 are vortexed once again atmaximum speed for 10 seconds. Denaturing occurs during the first andsecond water bath steps.

The final step is to transfer the prepared specimen for hybridization.In this step, the operator pipettes 75 microliters of the preparedspecimen into the bottom of an empty well in a hybridization microwellplate (e.g., a 96 well hybridization plate). After the microwell plateis loaded with specimens and calibrators or quality control samples, theplate is transferred to an automated or manual system for furtherprocessing to assess whether the sample is infected with a number ofdifferent HPV types (e.g., types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56,58, 59 and 68).

The foregoing HC2 protocol is just one example of a sample processingprotocol that is used in conjunction with a sample assay process. Otherprotocols, and particularly manual protocols for preparing a sample inanticipation of further processing or evaluation, are known in the art.In some cases, the protocol is a regulated protocol that is indicatedonly for particular uses, and should be followed as closely as possibleto maintain regulatory compliance.

Many sample processing protocols are specifically designed to beperformed partially or entirely by hand. In some cases, the manual stepscomprising the protocol may not be readily performed by an automatedsystem. For example, the foregoing HC2 sample preparation protocolincludes a number of steps particularly suited to manual operation(e.g., pellet observation, decanting, denaturing). These processes maynot be readily-amenable to automated processing of multiple samples.Furthermore, where the protocol is regulated, it may be difficult tosimulate the manual steps in an automated environment. Still further,even where a manual protocol is converted to an automated process, theremay remain a question of whether the two processes are truly comparable,as numerous innocuous-seeming deviations from the protocol that arerequired by the automated process may, in fact, substantially affect thefinal results.

The conversion of manual protocols to automated processes can presentmany challenges, and numerous unforeseen issues often arise. Such issuesrequire novel and unique solutions to ensure that the automated processis truly comparable to an existing manual protocol.

SUMMARY

In one exemplary embodiment, there is provided a method for visuallyinspecting pelletized samples. The method generates an inspection imageof the bottom of a sample tube holder. The sample tube holder has anumber of sample tube locations, and each sample tube location isconfigured to receive a sample tube in it. The inspection image isevaluated to determine whether a tube image exists for each sample tubelocation, and each sample tube image is evaluated to determine whether apellet is located in each sample tube.

The recitation of this summary of the invention is not intended to limitthe claims of this or any related or unrelated application. Otheraspects, embodiments, modifications to and features of the claimedinvention will be apparent to persons of ordinary skill in the art inview of the disclosures herein.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the exemplary embodiments may be understood byreference to the attached drawings, in which like reference numbersdesignate like parts. The drawings are exemplary and not intended tolimit the claims in any way.

FIG. 1 is a front view of an exemplary sample preparation apparatus.

FIG. 2 is a schematic plan view of an exemplary processing module.

FIG. 3 is a flowchart of exemplary process steps for operating a sampleprocessing module.

FIG. 4 is a schematic plan view of another exemplary processing module.

FIG. 5 is a flowchart of exemplary process steps for operating a sampleprocessing module.

FIG. 6 is a schematic plan view of another exemplary processing module.

FIG. 7 is a schematic plan view of another exemplary processing module.

FIG. 8 is a cutaway side view of a first exemplary ultrasonic sampleadequacy detector.

FIG. 9 is a cutaway side view of a second exemplary ultrasonic sampleadequacy detector.

FIG. 10 is a schematic representation of a first ultrasonic backscattersignal.

FIG. 11 is a schematic representation of a second ultrasonic backscattersignal.

FIG. 12 is a schematic representation of a third ultrasonic backscattersignal.

FIG. 13A illustrates an exemplary decanting system shown in the readystate prior to decanting.

FIG. 13B illustrates an exemplary decanting system shown in thedecanting position.

FIG. 14 illustrates a pair of exemplary decanting grippers, with oneshown in the upright position and the other in the Inverted position.

FIG. 15 is an exploded view of an exemplary decanting gripper of FIG.14.

FIG. 16A illustrates an exemplary tube strip being held in an uprightposition by the decanting grippers of FIG. 14.

FIG. 16B illustrates an exemplary tube strip being held in an invertedposition by the decanting grippers of FIG. 14.

FIG. 17 illustrates an exemplary embodiment of a tube strip.

FIG. 18 illustrates a pair of exemplary transport grippers.

FIG. 19 illustrates the transport grippers of FIG. 18 holding the tubestrip of FIG. 17.

FIG. 20 is a schematic plan view of an exemplary blotting system.

FIG. 21 illustrates an exemplary blotting system with an inverted tubestrip prepared for blotting.

FIG. 22 is a view of an exemplary disposal arm.

FIG. 23A is a front view of an exemplary sheet gripper, shown in theopen position.

FIG. 23B is a front view of an exemplary sheet gripper, shown in theclosed position.

FIG. 24A is a partially cut away side view of an exemplary disposal arm,shown in the open position.

FIG. 24B is a partially cut away side view of the disposal arm of FIG.24A, shown in the closed position.

FIG. 25 is a cross sectional view of the lower end of the disposal armof FIG. 24A, as viewed in the plane of the disposal arm's arm axis.

FIGS. 26A-D illustrate four operating positions of the disposal arm ofFIG. 24A.

FIGS. 27A-C illustrate an exemplary process for picking up and removingused sheets from an exemplary blotting sheet supply.

FIG. 28 is an exploded view of an exemplary heating assembly.

FIG. 29 is a lateral cross sectional view of the heating assembly ofFIG. 28, shown in the fully-assembled configuration, and with the crosssection taken through the axis of a tube strip.

FIG. 30 is a lateral cross sectional view of the heating assembly ofFIG. 28, shown in the fully-assembled configuration, and with the crosssection taken through a lateral wall of the tube strip holder.

FIG. 31 is a longitudinal cross sectional view of the heating assemblyof FIG. 28, shown in the fully-assembled configuration.

FIG. 32 is a schematic temperature versus time plot comparing a manualheating process to an automated heating process.

FIG. 33 is a cross sectional view of an exemplary cover, tube and tubestrip holder, as viewed through a downwardly-extending protrusion.

FIG. 34 is an exemplary light exposure of one embodiment of a tube stripholder.

FIG. 35 is an exemplary embodiment of a tube strip holder formed as anoptical mask.

FIG. 36 illustrates exemplary centrifuge loads on an exemplary tubestrip holder, tube strips and centrifuge bucket.

FIG. 37 is a cut away isometric view of an exemplary vision inspectionsystem.

FIG. 38 is a perspective silhouette image of a tube strip holder, tubestrips and samples, as viewed from the bottom.

FIG. 39 is a flowchart of an exemplary process for identifying regionsof interest in an image such as shown in FIG. 38.

FIG. 40 is a representative image of the regions of interest and otherinformation extracted from the image shown in FIG. 38, using the processof FIG. 39.

FIG. 41 is an enlarged view of the four tubes located at the center ofthe bottom two rows of tubes shown in FIG. 40.

FIG. 42 is a flowchart of an exemplary process for determining thepresence of a pellet in an image such as shown in FIG. 38.

FIG. 43 is a graphic illustration of steps in an exemplary process foranalyzing the dimensional properties of a particle to determine whetherit represents a sample pellet.

DETAILED DESCRIPTION

The exemplary embodiments described herein relate to automated samplepreparation apparatus and methods, and an exemplary embodiments relateparticularly to apparatus and methods used to automate the manual samplepreparation steps associated with the HC2 protocol. However, it will beunderstood that embodiments of the invention can be used to prepareother kinds of sample. For example, the combination of pipetting,mixing, incubation, centrifugation, and decanting is applicable to DNAextraction, concentration, and purification as a front end toenzyme-linked immunosorbent assays (“ELISA”), non-amplified probetagging or sandwich assays, and target amplification assays. Theexemplary processing modules described herein may be readily modified toinclude additional equipment that might be necessary to performalternative processes. For example, a processing module may bereconfigured to include magnetic bead binding, plate washing, andmultiple optical detection devices.

Referring to FIG. 1, an exemplary embodiment of an automated samplepreparation apparatus 100 is illustrated. In general terms, theapparatus 100 includes a processing module 102 that may be mounted on astand 104. The processing module 102 may contain some or all of theoperating parts, storage facilities for supplies, and so on. The stand104 may include additional components, such as a power supply, reagentsupplies, consumable supplies, and the like. The stand 104 also mayprovide a mounting point to align and attach external components thatare part of the processing module 102 (e.g., computer hardware,centrifuges, vision systems, etc.). The stand 104 may be incorporatedinto the processing module 102 to form a large processing module 102,but alternatively the processing module 102 may be operationallyindependent of the stand 104 so that the processing module 102 can beused as a table-top unit. The processing module 102 preferably includesa housing 106, having one or more openable covers, such as a transparentfront panel 108. Suitable lockout systems may be provided to preventoperation when the housing 106 is open. The apparatus 100 may alsoinclude a computer processing unit, which may be integrated into theprocessing module 102, located in a remote or separate processor such asan external computer 110, or distributed over a network of communicatingprocessors. The computer processing unit may be operatively connected toa variety of robotic devices located in the processing module 102, suchas pipettors 112, transport mechanisms, heaters, optical equipment,shakers, barcode readers, and the like.

Referring to FIG. 2, an exemplary processing module 102 is illustratedin schematic plan view. The processing module 102 may be assembled, inwhole or in part, on a flat platform 200 comprising a number ofuniformly-shaped and uniformly-distributed lanes 202 that extend fromthe front 204 of the module 102 to the back 206 of the module (forclarity, only some of the lanes 202 are marked by reference number 202).The lanes 202 may include physical dividers, mounting racks, mountingholes, and other features to permit installation, removal, replacement,and possibly rearrangement, of various working parts of the processingmodule 102. Such automated system platforms 200 are known in the art andneed not be described further herein.

The processing module 102 of FIG. 2 is configured for automatedprocessing of samples in an automated equivalent to the manual HC2protocol. However, other uses are envisioned for this or otherconfigurations of a processing module 102.

The processing module 102 includes a number of tube strip racks 208 inwhich tube strips 210, such as the embodiments described subsequentlyherein, are provided. Each tube strip 210 includes a plurality of tubes,and each tube may contain a separate specimen for processing. Tubestrips 210, or individual tubes in a tube strip 210, also may containcalibrators or quality controls (or no sample). For purposes of thisdescription, the contents of each tube will be considered to be a“sample” regardless of whether its contents are a patient's specimen, acalibrator, a quality control, or nothing at all. In this embodiment,each tube strip 210 has four tubes, each tube strip rack 208 can hold upto six tubes strips 210. Nine tube strip racks 208 are illustrated, buteight racks 208 or other numbers of racks 208 may be provided. The tubestrip racks 208 may be provided in adjacent lanes 202, or otherwisedistributed.

Tube strip holders 212, such as those described subsequently herein, arealso provided in the processing module 102. The tube strip holders 212are configured to hold one or more tube strips 210 for transport throughthe processing module 102, and during further processing steps. In thisexample, there are eight tube strip holders 212, and each can hold sixtube strips 210. Thus, each tube strip holder 212 can hold up totwenty-four samples. The tube strip holders 212 may be provided next to,or on either side of, the tube strip racks 208. For example, four tubestrip holders 212 may be provided on each of two platforms mounted tothe lanes 202 located on either side of the tube strip racks 208.

The processing module 102 also may include one or more mixing devices,such as orbital shakers 214. The orbital shakers 214 may include aheater or chiller to heat or cool the samples in the tubes. The orbitalshakers 214 or other mixing device may be any device suitable for mixingthe contents of the tubes, and preferably is able to mix the tubeswithout removing them from the tube strip holders 212. For example, theorbital shakers 214 may be Hamilton Heater Shakers available fromHamilton Robotics of Reno, Nev. The orbital shakers may be operated atroom temperature (i.e., no heating), or at a predetermined temperaturecontrolled by heating elements coupled to the shakers (e.g., aresistance coil or the like). Multiple orbital shakers 214 may beprovided for simultaneous, separate, processing of multiple tube stripholders 212. The shown orbital shakers 214 each hold a single tube stripholder 212.

One or more centrifuges 216 may be provided in or with the processingmodule 102. In the exemplary embodiment, a centrifuge 216 is providedadjacent one end of the platform 200. The centrifuge 216 may be mountedon the platform, but (like the other components described herein) thisis not required. In the shown embodiment, the centrifuge 216 isconnected to the processing module 102 independently of the platform200, which may assist with supporting the large weight of the centrifuge216, and isolating the centrifuge 216 during operation. An example of acentrifuge that may be used is the HiG™ centrifuge available from BioNexSolutions, Inc., of Sunnydale, Calif.

The processing module 102 also may include one or more vision inspectionstations 218. The vision inspection station 218 may be located in acamera enclosure 220 having one or more cameras and one or more lightsources. If necessary, the camera enclosure 220 may be closable toprevent ambient light from interfering with the procedures carried outby the vision inspection station 218. One exemplary vision inspectionstation 218 may be provided in the STAR Line of products from HamiltonRobotics of Reno, Nev. Other sample inspection devices, such asturbidity meters and the like, may be used in other embodiments.

A decanting station 222 also may be provided in the processing module102. The decanting station 222 may comprise a system adapted to decantfluid from the sample tubes. An exemplary embodiment of a decantingstation 222 is described in detail subsequently herein. As shown in FIG.2, the decanting station 222 may include a retainer 224 to hold a tubestrip holder 212 while individual tube strips 210 are removed anddecanted. The decanting station 222 also includes a decant waste well226 to receive the supernatant decanted from the tubes. The decantingstation 222 may also include a gripper parking location 228 to receivegrippers used in the decanting process or other processes.

A tube strip lid holder 230 may be provided to hold a supply of one ormore tube strip covers, such as those described subsequently herein. Ifa separate gripper or gripping tool is used to manipulate the tube stripcovers, it may be stored in a cover gripper parking location 232.

The processing module 102 may include one or more liquid supplies, suchas reagent reservoir racks 234. The reagent reservoir racks 234 may beslidably mounted on the platform lanes 202 for ease of removal andrefilling. The reagent reservoir racks 234 may include any reagent orcombination or reagents used for the processing steps conducted by theprocessing module 102. For example, each reagent reservoir rack 234 mayhave five 50 milliliter reservoirs. The contents of the reservoirs mayvary, and may include transport media, denaturation reagents, sampleconversion buffers, and the like. In other embodiments, liquid suppliesmay be provided in bottles or other containers located elsewhere inoperative association with the processing module 102. For example,liquid supplies may be provided in the stand 104 and connected by hosesto dispensers (not shown) in the processing module 102.

Pipette tip storage units 236, 238 may be provided to supply disposablepipette tips. In the example shown, there are two 5-milliliter pipettestorage units 236 that each hold 24 tips, and eight 1-milliliter pipettestorage units 238 that each hold 96 tips. Other numbers and variationsof pipette storage arrangements may be used in other embodiments, andthese may be omitted if they are not necessary, or replaced with otherdisposable supply units.

The processing module 102 also may include heating units 240, such asheated water baths or the like. The heating units 240 may be configuredto heat the individual tube strips 210, individual tubes (if usedinstead of tube strips 210), or tube strips 210 that are mounted in tubestrip holders 212. In a preferred embodiment, the heating units 240 mayhave heat distribution features configured to help uniformly heat tubestrips 210 that are mounted in tube strip holders 212. One example of aheat distribution feature is described subsequently herein, butvariations of the shown example, or other devices, may be used in otherembodiments of processing modules 102. If desired, the heating units 240may also be equipped to mix the contents of the tubes during the heatingprocess. For example, the heating units 240 may comprise Hamilton HeaterShakers available from Hamilton Robotics of Reno, Nev. Such units may bemodified or used in their original form.

The processing module 102 may be configured to hold one or more outputplates 242, such as typical 96-well plates. The output plates 242 may beused to store processed samples, or to hold the samples in advance offurther processing. For example, the sample plates 242 (which aresometimes be referred to as “hybridization” plates) may be configuredfor subsequent manual or automated performance of additional steps tocomplete the HC2 protocol. In the embodiment of FIG. 2, the outputplates 242 may be mounted on a sliding rack 250 along with one or more(In this case, four) tube strip holders 212. This allows simultaneousloading of tube strip holders 212 and output plates 242. Conveniently,the parts may be arranged so that the number of samples that are held inthe tube strip holders 212 equals the number of wells in the outputplate (in this case, the number is ninety-six). In this way, the entirerack 250 can be removed when processing is complete, and reloaded withempty tube strip holders 212 and an output plate 242 to quickly andefficiently prepare for a new processing run.

The output plates 242 may be covered by a lid. The lids may be providedwith the output plates 242 when the output plates 242 are loaded intothe processing module 102, or a supply of lids may be provided in theprocessing module 102. The processing module 102 may include a lidholder 244 that holds a supply of lids, or provides a place to storeeach output plate's lid as the output plate 242 is being filled.

The processing module 102 may include other features, such as a tipeject station 246 to receive used pipette tips, or other waste chutes orcontainers for disposables. Another feature may be an additional tubestrip rack 248 that holds additional empty tube strips 210. The emptytube strips 210 may be placed in the tube strip holder 212 to balancethe centrifuge 216 if a tube strip holder 212 is processed without acomplete set of sample-filled tube strips 210.

The various samples containers, consumables and supplies may be loadedby opening the cover 108 or other access doors. However, in a morepreferred embodiment, at least some of these may be accessible throughone or more uncovered openings. For example, the tube strip racks 208and reagent reservoir racks 234 may be accessed through an opening underthe bottom edge of the cover 108, to permit removable and replacement toallow continuous resupply during operation.

A suitable robotics system may be provided in the processing module 102and configured and programmed to conduct the processing steps describedherein. For example, automated pipetting and material handling systems,such as the pipettors, autoloaders, iSWAP microplate grippers, and CO-REgrippers in the STAR Line of robotics provided by Hamilton Robotics ofReno, Nev., may be used to pipette fluids and transport tube strips 210and tube strip holders 212 throughout the processing module 102. Pipettecontrol systems that use one or more sensors or algorithms to detectclots or otherwise monitor aspiration properties also may be used forany of the pipetting steps described herein. Other features, such assafety locks, lights, ventilation or seals, consumable supplies, and thelike, may be included in or with the processing module 102, as desiredfor the particular application.

It will be understood that the various components may be rearranged,such as by swapping locations or stacking them on vertically-displaceddecks. Other embodiments also may use different numbers of processingstations and different numbers of supplies and samples, and the like.Other variations and modifications will be apparent to persons ofordinary skill in the art in view of the present disclosure.

FIG. 3 illustrates an exemplary process that may be performed, at leastin part, by a processing module 102 such as the exemplary embodiment ofFIG. 2. In this embodiment, the process is an automated equivalent of amanual HC2 processing protocol. Before the process begins, an operatormay manually add 5 drops of indicator or dye to a denaturation reagent(“DNR”) causing the DNR to turn dark purple. This is the same as theconventional manual HC2 protocol. At step 300, the operator resuspendsone or more samples provided in individual containers (e.g., samplecollection tubes), either by hand or by using a conventional table-topvortex mixer. The samples may be provided in any kind of sample vial orcontainer. In the case of HPV testing, individual patient samples areoften provided in PreservCyt® vials available from Hologic, Inc. ofMarlborough, Mass. Such PreservCyt® vials are commonly used to collectcervical samples, and each vial includes a sample from a single patientand a preservative liquid. Other common sample formats include theSurePath™ format from Becton, Dickinson and Company of Franklin Lakes,N.J., and others.

Next, in step 302, the operator manually aliquots a 4 milliliterspecimen from each sample into a respective tube of a tube strip 210.This manual step may be done using the same method that the HC2 protocolindicates for manually transferring the sample to a sample processingcontainer. Next, in step 304 the operator loads the filled tube strips210 into the tube strip racks 208, and loads the tube strip racks 208into the processing module 102. In the embodiment of FIG. 2, theoperator can load enough tube strips 210 to fill two 96-well outputplates 242, but other capacities may be used. The operator may continueloading tube strips 210 after processing begins, in order to provideenough samples to fill additional output plates 242. Steps 300 and 302may be performed manually, but in other embodiments some or all of thesesteps may be performed by another processing system that operatesoutside the processing module 102, or in the processing module 102itself.

Next, in step 306, the processing module 102 transports the tube strips210 to a tube strip holder 212. The tube strips 210 may be manipulatedby any suitable transport mechanism, such as the transport grippersdescribed subsequently herein. It will be appreciated that step 306 mayalternatively be performed after step 308 described below, in which casethe Sample Conversion Buffer (“SCB”) would be dispensed into the tubestrips 210 before they are loaded into the tube strip holder 212.

In step 308, the processing module 102 activates one or more pipettors,such as a ganged group of four or eight pipettors, to withdrawconventional HC2 protocol SCB from the reagent reservoirs 234, anddispense the SCB into the samples contained in the tube strips 210. Theprocessing module 102 can operate the pipettors and other devices usingany control system, such as an internal or external processor. Inkeeping with the manual HC2 protocol, 400 microliters of SCB may bedispensed into each tube, but other volumes may be dispensed in otherembodiments. The pipetting process may also include moving the pipettorsto a disposable pipette tip supply, such as a 5-milliliter pipettestorage unit 236, moving the pipettor to engage disposable tips beforewithdrawing the SCB, and moving the pipettor to deposit the used pipettetips into the tip eject station 246 after dispensing the SCB. In otherembodiments, this or other fluid deposition steps may be performed bydispensers or other devices that are plumbed directly to fluidreservoirs, to minimize the need to use disposable pipette tips orincrease processing speed.

In step 310, each tube strip holder 212 is transported to an orbitalshaker 214, where the samples in the tubes are mixed. Any suitabletransport mechanism may be used for this step and for other transportsteps described herein. In an exemplary embodiment, the tubes are mixedat room temperature by operating the orbital shaker 214 for 30 secondsat 800 rpm on a 3 millimeter orbit.

Next, in step 312, the tube strip holder 212 is loaded into thecentrifuge 216 and centrifuged at 2,900 gravities for 15 minutes. Thecentrifuging step is expected to cause the samples in the tubes tocreate cell pellets at the bottom of each tube.

To verify that a pellet has formed in each tube, in step 314 theprocessing module 102 loads the tube strip holder 212 into the visioninspection station 218. Here, the tubes are visually inspected,preferably all at one time, to evaluate whether a pellet has formed ineach tube. In the shown embodiment, verification may be performed usinga camera below the tubes, and a light above the tubes, but a reversearrangement or other arrangements or detecting devices may be used. Ifthere is no pellet, a record is made of the tube lacking a pellet, andthe process continues. The visual image generated in step 314 may beprinted or electronically stored for future reference. Step 314 may beomitted in an alternative embodiment. For example, the HC2 protocolcalls for checking the presence of the pellet after centrifuging, butdoes not necessarily modify the remaining process depending on theoutcome of this inspection; thus, for an automated version of the HC2protocol, step 314 may optionally be omitted.

In step 316, the tube strip holder 212 is transported to the decantingstation 222, where it is placed on the retainer 224 for storage duringthe decanting process. In the exemplary embodiment, the decantingprocess may be performed by sequentially removing each tube strip 210from the tube strip holder 212, inverting the tube strip 210 to decantthe supernatant from each tube into the decant waste well 226, turningthe tube strip 210 upright, and replacing it in the tube strip holder212. The tube strips 210 may be decanted by devices such as thosedescribed elsewhere herein, or by other mechanisms. For example, twopipette channels may be used to pick up decanting grippers from thegripper parking location 228, and the pipette channels and decantinggrippers may be used to rotate the tube strip 150 degrees to decant thefluid. The rotation speed, rotation angle, and decanting time may beadjusted to obtain the results desired for the particular application.In an alternative embodiment, the tube strips may remain in the tubestrip holder 212 during step 316, in which case the retainer 224 may beomitted. For example, the decanting process may be replaced with anaspiration step, in which the tube strips 210 remain upright, andsupernatant is removed by aspiration.

Immediately after decanting, it may be desirable to blot the tube strips210 to help remove any fluid that remains in or on the tubes. An exampleof a blotting system is described below. Such a process step is notrequired in all embodiments.

Following decanting and replacing the tube strips 210 to the tube stripholder 212, in step 318 the tube strip holder 212 is returned to thevision inspection station 218. In this step, the vision inspectionstation 218 is operated to verify that the pellet remains in each tube.If a pellet is missing, a record is made of the tube lacking the pellet,and the process continues. The visual image generated in this step maybe printed or electronically stored for future reference. Followinginspection, the tube strip holder 212 is moved to its original location(e.g., on sliding rack 250), or to an intermediate holding location.

Next, in step 320, the processing module 102 operates the pipettors toload pipette tips, such as disposable 1 milliliter tips from one of the1-milliliter pipette storage units 238, withdraw HC2 protocol SpecimenTransport Medium (“STM”) from the reagent reservoirs 234, dispense theSTM into the samples contained in the tube strips 210, and eject theused pipette tips into the tip eject station 246. In one embodiment, avolume of 100 microliters of STM is dispensed into each tube, but othervolumes may be used in other embodiments.

In step 322, the processing module 102 essentially repeats step 318, butin this step it withdraws HC2 protocol Denaturation Reagent (“DNR”) fromthe reagent reservoirs 234, and dispenses the DNR into the samples inthe tube strips 210. In one embodiment, a volume of 50 microliters ofDNR is dispensed into each tube, but other volumes may be used in otherembodiments. Steps 318 and 320 may be reversed or performedsimultaneously in other embodiments.

In step 324 the tube strip holder 212 is moved to a room-temperatureorbital shaker 214 to resuspend the pellets in the tubes. The orbitalshaker 214 is operated for two minutes at 1,250 rpm on a 3 millimeterorbit. If necessary, a cover, such as the covers described below, may beplaced on the tube strip holder 212 prior to this mixing step to preventcross-contamination during the mixing process.

Next, in step 326, the tube strip holder 212 is moved to a heater-shaker240 and the samples in the tube strip holder 212 are heated andperiodically mixed. If a cover is not already on the tube strip holder212, one may be installed prior to heating. In an exemplary embodiment,the samples are heated to 65° (±2°) Celsius for 15 minutes, mixed (whilestill heating) at 1,250 rpm on a 3 millimeter orbit for 30 seconds,heated for another 30 minutes, and mixed at 1,250 rpm on an orbit of 3millimeters for 10 seconds. The target temperature is maintained for theentire heating and mixing process, yielding a total incubation time ofapproximately 45 minutes and 40 seconds.

In step 328, the tube strip holder 212 is transported to its originallocation, such as the sliding rack 250, or moved to an intermediateholding station. The processing module 102 removes the cover from thetube strip holder 212 using any suitable gripper or transport mechanism,and stores or disposes of the cover. In order to preventcross-contamination caused by condensation or liquid clinging to thecover, the cover may be removed by lifting it straight up from the tubestrip holder 212. The processing module 102 also removes the lid fromthe output plate 242 (if one is provided) and places it on the lidholder 244. Next, the pipettors pick up tips (e.g., 1-milliliter tips)and transfer 75 microliters of sample from each tube to an empty well inthe output plate 242. It may take several tube strip holders tocompletely fill the output plate 242. For example, one output plate 242may receive samples from four tube strip holders 212. Some of the outputplate 242 wells may be loaded with controls or other non-samplecontents. Once the output plate 242 is filled or deemed to besufficiently filled for further processing, the lid is replaced on theoutput plate 242. Once it is filled, the output plate 242 may beimmediately removed from the processing module 102, or stored in theprocessing module 102 for a period before it is removed.

To reduce processing times, the processing module 102 may have multipleessentially identical processing devices or equipment that are operatedat the same time to process different samples. For example, theprocessing module 102 may have four heater-shakers 240 to process fourtube strip holders 212 at the same time or in close succession.

The process described with reference to FIG. 3 may also include anynumber of intermediate steps, such as steps taken to track samples or toensure processing stability. For example, barcodes may be provided onthe tube strips 210 and tube strip holders 212, and these barcodes maybe read periodically to track sample locations. Also, the processingmodule 102 may be programmed to run a predetermined number of specimensas a batch to ensure that all necessary supplies and reagents areavailable to completely process all of the specimens withoutinterrupting the run. However, if the number of specimens is less thanthe available output volume (e.g., if 192 wells are available on twooutput plates 242, but there is only room to load 96 samples at a time),the processing module 102 may be programmed to allow more samples to beloaded during operation to fully utilize the output capacity.

FIG. 4 illustrates another exemplary processing module 400. Processingmodule 400 may have a number of elements in common with the embodimentof FIG. 2, and such parts are designated by the same reference number.The embodiment of FIG. 4 may be a deviation of the embodiment of FIG. 2,and in a preferred embodiment a single set of equipment can be assembledas shown in FIG. 2 or as shown in FIG. 4. The use of sliding racks andthe like may facilitate such conversion by allowing simple moving,removal and replacement of parts. Suitable reprogramming of theapparatus can be done along with the conversion, either automatically ormanually. For example, the processing module 400 may be switched by anoperator into an alternative, pre-programmed operating mode, or thereplacement parts may be coded (e.g., by radio frequency identificationtags or barcodes) to automatically indicate to the processing module 400that a change has been made and/or that reprogramming to a differentoperating mode may be required. If the system is configured to recognizethe parts and their locations, the system may automatically switchoperating modes upon recognizing a known arrangement of parts, and maytransmit an error message if the arrangement is not recognized.

The processing module 400 of FIG. 4 differs from the embodiment of FIG.2 primarily in the replacement of a number of tube strip racks 212 withvial racks 402. The vial racks 402, which may be racks that slide intothe platform lanes 202, are each configured to hold one or more samplevials 404. In this case, seven tube strip racks 212 are removed, andreplaced by four vial racks 402. In this example, each vial rack iscapable of holding up to twelve sample vials 404, yielding a maximum offorty-eight sample vials 404. Other numbers of racks and vials may beused in other embodiments. Other modifications, as compared to theembodiment FIG. 2, include the removal of the empty tube strip rack 248,and moving the right-hand sliding rack 250 to make room for the vialracks 402.

The embodiment of FIG. 4 facilitates a modified operation mode in whichsamples are provided to the system in individual sample vials 404. Thisis in contrast to the embodiment of FIG. 2, in which specimens areprovided to the apparatus pre-dispensed into tube strips 210. The samplevials 404 may be any suitable sample vial. For example, in the case ofHPV testing, the sample vials 404 may comprise one or more varieties ofPreservCyt® or SurePath™ vials.

The vial racks 402 may hold the sample vials 404 in any suitablefashion. For example, each vial rack 402 may have a plurality of vialwells in which the sample vials 404 are dropped before sliding the vialrack 402 into the processing module 400. Such wells may be vertical ortilted at an angle, such as an angle between 5° and 20° from vertical(e.g., 10° from vertical may be suitable for some typical sample vials404), to facilitate pipetting of the contents from the sample vials 404.The wells also may be mounted to tilt during the pipetting process. Thewells may have openings or slots through which barcodes on the sides ofthe sample vials 404 can be read before, during, or after insertion ofthe vial rack 402 into the processing module 400. The sample vials 404may be provided in the vial racks 402 with their caps removed, or adecapping/recapping unit may be integrated into the processing module400 to remove and replace the vial caps during processing. If desired,the vial racks 402 may include features, such as shakers or the like, tosuspend the samples.

Where individual sample vials 404 are processed by the processing module400, it may be desirable to provide features and systems to evaluate theadequacy of the sample for further processing or the need for correctiveprocessing. For example, a sample adequacy system in the form of aturbidity instrument or ultrasonic testing instrument 406 may beprovided to determine whether the sample has sufficient cell count towarrant further processing. A preliminary check of this sort can avoidunnecessary processing, which can save on reagent cost and processingtime, and also prevent the recording of a false negative result. Anexample of an ultrasonic sample adequacy detection system is describedsubsequently herein. This or other sample adequacy detection devices maybe incorporated anywhere in the processing module.

As noted above, processing module also may include pipette controlsystems to detect clots or otherwise monitor aspiration properties. Thismay be particularly desirable where the samples are provided as anoriginal, unprocessed biological sample, such as in a PreservCyt® orSurePath™ vial. Such samples may include tissue, clots, and otherfeatures that might inhibit proper pipetting.

FIG. 5 is an exemplary process for operating processing module 400. Instep 500, the operator vortexes (e.g., in a vortex mixer at its maximumsetting) or vigorously shakes each sample vial 404 to resuspend thesample. Next, in step 502, the operator loads each sample vial 404 intoa sample rack 402, and, if applicable, orients any barcode or othercomputer-readable indicator so that it can be read as the sample rack402 is inserted into the processing module 404. Next, in step 504 theoperator loads the sample rack 402 into the processing module 400. Afterprocessing begins, the operator may remove sample racks 404 that havebeen processed, and replace them with filled sample racks 404, toprovide continuous operation to process a large number of specimens. Theforegoing steps may be performed manually or by another processingsystem that operates outside the processing module 400.

In step 506, the processing module 500 operates the pipettors to loadpipette tips, such as 5-milliliter disposable pipette tips, andhydraulically mix the contents of each sample vial 404. Hydraulic mixingmay be performed, for example, by drawing a volume from of each samplevial 404 into the pipette tip expelling the volume back into the samplevial 404. This may be repeated one or more times to obtain the desiredmixing results. In alternative embodiments, shakers or other devices maybe used to mix the samples.

Following mixing, in step 508 the processing module 500 operates thepipettors to transfer a volume of each sample, such as 4 milliliteraliquot, to a respective tube in a tube strip 210. A clot detectionsystem or other algorithm may be used to determine whether sample isproperly pipetted, and such algorithm may include a process to retrypipetting if a failure condition is detected. If a failure is detectedand cannot be overcome, the processing module 400 may associate a recordof the failure with the particular sample vial 404. The same pipette tipthat was used for hydraulic mixing is used to transfer the sample. Usedtips are disposed in the tip eject station 246. If a clot is detected onthe outside of a pipette tip it may drip and contaminate the instrumentor samples. In this scenario the tip can be placed into the sample vial404 and left there to prevent cross-contamination. Steps 506 and 508 canbe performed simultaneously on a number of samples using a gangedpipettor. If adjacent sample tubes 404 on the sample rack 402 are spacedapart by a different distance than adjacent tubes in the tube strip 210,it may be desirable to use a pipettor assembly with variable channelspacing to accommodate the different spacings.

The foregoing process also may include manually adding 5 drops ofindicator or dye to the HC2 DNR causing the DNR to turn dark purple, asdone in the manual HC2 protocol. This step may be performed at any timeprior to the DNR being used in the remaining process steps. This stepalso may be automated into the processing module's functions.

Following step 508, the tube strips 210 are processed, such as describedabove, to convert the specimens and transfer them to output plates 242.For example, step 508 may be followed by step 305 (dispensing SCB intothe tube strips 210), and the remaining steps until the specimens aretransferred to plates in step 328.

FIG. 6 shows another embodiment of a processing module 600. Thisembodiment may be a separate construction, or a modification of theearlier-described processing modules 102, 400. The embodiment of FIG. 6is configured to start the process with samples provided in tube strips210 that are already loaded into tube strip holders 212. For example,the tube strip holders 212 may contain tube strips 210 that werepreviously processed through step 326 of FIG. 3 by a processing module102 as shown in FIG. 2, and then stored (e.g., refrigerated or frozen)prior to step 328 of FIG. 3. In this embodiment, the processing module600 is used to perform step 328 of FIG. 3—that is, to transfer theprocessed samples from the tube strip holders 212 to output plates 242.The arrangement is expected to be helpful to schedule more efficientoverall processing times where large volumes or interruptions (e.g., theend of a working shift) are expected to occur.

The embodiment of FIG. 2 may be configured as shown in FIG. 6 byremoving all of the tube strip racks 208 (Including the rack of emptytube strips 248) and reagent reservoirs 234, and installing two or moresliding racks 250 holding tube strip holders 212 and output plates 242.Alternatively, the tube strip racks 208, 248 and reagent reservoirs 234may remain in place and left unused during processing.

FIG. 7 shows another alternative embodiment of a processing module 700that is set up for rapid retesting preparation. This embodiment also maybe a permutation of the foregoing processing modules 102, 400, 600, or aseparate construction. Here, the reagent reservoirs 234, tube stripracks 208, 248 and vial racks 402 are omitted, and replaced by slidingracks 250. One sliding rack 250 holds an output plate 242 and four tubestrip holders 212, and four additional sliding racks 250 hold more tubestrip holders 212. This arrangements increases, and may maximize, thenumber of tube strip holders 212 that are held by the apparatus withoutrequiring substantial modification. It may be necessary to remove othercomponents, such as the tube strip lid holder 230, if access to the tubestrip holders 212 is impeded. To this end, the tube strip lid holder 230and other parts may be provided on one or more sliding racks 702.

The embodiment of FIG. 7 is operated to transfer specimens from samplesthat require retesting to one or more output plates 242. Since (at leastin the case of HC2 testing) the percentage of samples requiringretesting can be very small, the retest candidates may be distributedamong a large number of tube strip holders 212. As such, it may behelpful to provide a large number of tube strip holders 212 as comparedto the number of output plates 242. In the shown example, twenty-fourtube strip holders (up to 576 individual samples) are provided with assingle 96-well output plate 242, but other arrangements may be used, asdesired.

The embodiment of FIG. 7 is operated by providing the processing module700 with an electronic record identifying which tubes on which tubestrip holders 212 contain samples to be retested. The record may belinked to a barcode on each tube strip holder 212, or providedotherwise. As the sliding racks 250 are installed, a bar code reader(not shown) may read a barcode on each tube strip holder 212, providingthe processing module 700 with sufficient information to identify theexact location of each sample requiring retesting. After the slidingrack 250 is installed, the processing module 700 transfers a specimenfrom each sample to be retested from the tube strip holder 212 to theoutput plate 242. To facilitate retesting a large number of samples, theprocessing module 700 may transfer samples from the tube strip holders212 on the rightmost sliding rack 250, and progressively work left aseach sliding rack 250 is completed. Using this method (or other methodsin which each rack 250 is completely processed before moving to the nextrack 250), the operator can remove sliding racks 250 that have completedprocessing, and replace them with unprocessed racks 250, even while theprocessing module continues to transfer samples from other racks 250 tothe output plate 242. A suitable system of locks or indicator lights maybe provided to indicate which racks 250 are complete and ready forreplacement, and to prevent errant removable of racks 250 that have notbeen processed. Of course, the foregoing arrangement may be modified.For example, the individual tube strip holders 212 may be removed afterall samples to be retested have been transferred to the output plate242, and replaced with another tube strip holder 212.

Other variations on the layout and function of a processing module willbe readily apparent from the foregoing disclosure. For example, theembodiment of FIG. 6 can be modified to start with tube strip holders212 holding tube strips 210 that have been processed up through step 308of FIG. 3, and then stored or briefly retained for later processing. Inthis modification, the empty lanes 202 may contain reagent racks 233 toprovide the necessary reagents to complete the process of FIG. 3. Alsoin this modification, step 306 (dispensing SCB) may be performed as thefirst step, and step 308 would be omitted as being moot.

It will also be appreciated that any suitable sample tracking system maybe used with embodiments of a processing module. For example, barcodesmay be used to identify individual samples, associate groups of sampleswith particular tube strips 210, tube strip holders 212, and outputplates 242, and so on. Processing errors and other information may beimmediately associated with an electronic record for each sample byreference to the barcode-related information. Suitable barcode readersmay be used throughout the processing module to track progress andconfirm identity and process integrity at periodic intervals orlocations. Radio-frequency identification devices and other indicia mayalso be used in other embodiments.

Exemplary Sample Adequacy Modules

As noted above, it may be desirable to include a sample adequacyevaluation system in a processing module, particularly where processinga sample that lacks sufficient cells to provide a meaningful resultmight be expensive or risk reporting an erroneous test result. Forexample, a sample adequacy evaluation system may be desirable to detectthe presence of a sufficient number of epithelial cells to conductmeaningful tests for HPV. Sample adequacy evaluators, such asnephelometers and turbidity meters, have been used in a variety ofclinical settings, and devices that are compatible with automatedprocessing may be used with embodiments of the processing modulesdescribed herein. It has also been found that sample adequacy, and avariety of other sample properties, can be determined using sound waves,and particularly ultrasonic sound waves.

An exemplary embodiment of an ultrasonic sample adequacy detector isshown in FIG. 8. The detector includes a receptacle 800 having anultrasonic emitter or transducer 802 mounted to the bottom of thereceptacle 800. The transducer 802 is oriented to direct ultrasonicwaves in a vertical direction, and may comprise a conventional flat orfocused transducer. Any suitable transducer, such as a conventionalpiezoelectric transducer, that is capable of transmitting and detectingan ultrasonic wave may be used. Piezoelectric transducers are availablefrom a variety of sources, such as Olympus NDT of Center Valley, Pa.Exemplary equipment for use in this application may include aPanametrics 10 MHz 0.25 inch diameter element, with input providedthrough a programmable pulse generator and output fed through aprogrammable amplifier/filter and displayed on an oscilloscope.

The illustrated transducer 802 is mounted in a port through the bottomof the receptacle 800, but it may be located inside or below thereceptacle 800, or the bottom of the receptacle 800 may form a part ofthe transducer itself. If necessary, the exact location of thetransducer 802 may be adjusted to provide clearer or more focusedsignals. It will be understood that transducers are a combinedultrasonic transmitter and ultrasonic receiver, and the transducersdescribed herein may be replaced by separate transmitters and receivers.For brevity, references herein to a transducer will be understood toinclude an alternative transmitter/receiver pair.

The receptacle 800 is shaped and sized to receive one or more varietiesof sample vials 404. The embodiment of FIG. 8 receives a conventionalflat-bottomed vial 804, such as a PreservCyt® vial. If the transducer802 does not contact the bottom of the vial 804 sufficiently to reliablyconduct ultrasonic waves, it may be necessary to include a couplingmedium 806 to form an ultrasonic-transmitting pathway. The couplingmedium 806 may comprise a viscous or fluid compound, such as grease or aliquid bath, or a solid compound, such as an elastomeric compound.Combinations of the two may be used as well. To help ensure a propersound-transmitting path is formed, the tube 804 may be pressed into thecoupling medium 804. For example, a robotic arm that places the tube 804in the receptacle 800 may press the tube down into the coupling medium806 with a slight force during the sample adequacy testing process, or aseparate press or clamp may be provided to press the tube 804 intoplace.

The transducer 802 is configured to produce an audio (compressed waive)signal preferably at ultrasonic frequencies (above about 20,000 Hz). Theexpected scattering signal can be modified by altering the signalfrequency. For example, frequencies having a wavelength (the reciprocalof the frequency) that is much larger than the cell size are expected toproduce so-called Rayleigh scattering, and frequencies with a wavelengththat is much smaller than the cell size should produce specularscattering. Frequencies with wavelengths that are closer to the cellsize should produce intermediate scattering signals. For cells having asize of 60-100 micrometers, a wavelength of about 100 micrometers may beused to obtain intermediate scattering. Increasing wavelength couldprovide more diffuse Rayleigh scattering, but at the expense of overallintensity. Where the volume of the scattering media (cells) is less thanabout 20% of the total volume of the substance being tested, theintensity of the scattered signals is expected to be approximatelylinearly proportional to the scattering concentration. This is the casein testing typical epithelial cell concentrations in the context of HPVtest protocols, which often have a cell concentration of 1,000 cells permilliliter of preservative fluid, or less.

Any suitable control equipment may be used to operate the transducer totransmit and detect signals. Suitable devices and software programs arereadily available from transducer suppliers.

FIG. 9 shows an alternative embodiment, in which the an ultrasonicsample adequacy test receptacle 900 is configured to receive a taperedtube 904, but the remaining components are essentially the same as theembodiment of FIG. 8. Where a tapered tube 904 is processed, theultrasonic transducer 902 may be connected to the tube 904 by acontoured coupling medium 906 that cups the end of the tube 904 toprovide a larger contact area.

The sample adequacy test is performed by placing the tube 804 in thereceptacle 800, activating the transducer 802 to transmit a sourceultrasonic signal, and using the transducer 802 to detect the reflectedultrasonic signals (“backscatter”). (This description references theembodiment of FIG. 8, but it is equally applicable to the embodiment ofFIG. 9 and other embodiments.) The ultrasonic signal may comprise asingle event, but more preferably is a series of pulsed signal events toprovide better sampling data. The signal travels vertically through thecoupling medium 806 and the bottom of the tube 804, and into the liquidmedia 808 inside the tube 804. As the signal propagates upwards throughthe media 808, any significant changes in density will reflect a portionof the signal, and such reflected signals may be detected by thetransducer 802. For example changes in density may be caused when thesignal passes from one medium to another (e.g., passing from thecoupling medium 806 to the tube 804 and from the tube 804 to the liquid808), when the signal contacts a cell, or when the signal reaches theupper extent of the liquid 808 and passes into the air. If the densityof the cells are similar to the density of the liquid 808, it may benecessary to modify the density of one or the other to help generatereflected signals. The backscatter is also a function of therelationship between the sound wavelength and the particle (cell)diameter and an optimum frequency can be selected to maximize thereturn. The transducer 802 detects these reflected backscatter signals,and they are recorded by a computer operating the transducer 802.

A schematic representation of a backscatter signal is shown in FIG. 10,which is a plot of backscatter amplitude (A) versus time (t) for asingle ultrasonic pulse. FIG. 10 shows the backscatter signal as itmight appear. The transducer 802 begins recording at t₀, at which timethe acoustic backscatter begins to reflect back to the transducer 802.Time t₁ represents the time at which the signal reflecting off the uppersurface of the fluid reaches the transducer 802. The curve between t₀and t₁ indicates the distribution of the amplitude of the backscattersignal. Repeated pulses can be overlaid over this curve to enhancesampling resolution. It is expected that this backscatter signal can beused to detect a variety of sample properties.

A first sample property that can be evaluated using the backscatter datais the liquid level. Typically, the last large backscatter signal willbe generated as the ultrasonic signal reaches the meniscus of the liquid808 in the tube 804 and propagates into the surrounding air. The signalrapidly degrades after it leaves the liquid 808, and little or none ofthe signal will reflect back into the liquid 808 to be detected by thetransducer 802. The liquid level can be determined based on the totalamount of time it takes for the backscatter signal to reach thetransducer 802. For example, if t₁ is at or above a certain time value,then the liquid level may be considered a passing level. Suchqualitative assessments can be calibrated to the particular kind of tube804 and liquid media inside the tube, as well as other systemproperties, such as the type of coupling medium 806 and so on.Alternatively, the liquid level can be approximated numerically byconsidering the known dimensions of the different parts of the signalpath (i.e., the coupling medium 806 and tube 804) and the speed of soundin these various parts of the path. If the speed of sound is known forall of the path elements (coupling medium 806, tube 804 and liquid 808),then the maximum signal reflection signal time can be used, along withthe known dimensions, to calculate the depth of the liquid 808. Thedetails of this calculation will be readily apparent to persons ofordinary skill in the art, and need not be discussed here.

If the ultrasonic test reveals that the liquid level (and hence volume)is insufficient to perform a meaningful test, the sample may be rejectedand not subjected to further processing. Other variations on using thesignal to evaluate the liquid level will be apparent to persons ofordinary skill in the art based on the present disclosure.

A second sample property that can be evaluated using the backscatterdata is the cell concentration. Cells suspended in the liquid 808 willcause small backscatters at various times, depending on the height ofthe cell in the tube 804. Such backscattering typically appears (in highresolution plots and without smoothing), as grass-like noise between t₀and t₁. As the cell concentration increases, the amplitude of this noiseincreases. If the cells are generally equally distributed throughout theliquid 808, the noise will be generally equally distributed throughoutthe backscatter plot. A schematic example of this kind of evendistribution is shown in FIG. 10. The adequacy of the sample or anestimate of cellular concentration can be determined by comparing theproperties (e.g., average amplitude) of the backscatter to knownexamples, and a plot of the same can be readily developed usingempirical testing.

In contrast, FIG. 11 shows a schematic representation of a sample havinglittle or no cellular content, as indicated by the lack of scatteredreflections before the signal reaches the meniscus at t₁. The samplegenerating the plot in FIG. 11 may have insufficient cellular content tobe processed without risking a false negative result. In situations inwhich useful information about the cell concentration can be determinedby evaluating the settling rate of the cells, the transducer can beoperated multiple times (e.g., several times per second) to yieldcomparative backscatter data to indicate how rapidly the cells settleafter being suspended in the liquid 808.

A third sample property that may be evaluated from the backscatter datais the presence of foreign objects. Biological samples and other samplesare often collected and placed into sample tubes using swabs, brushes,sponges, and other collection implements 810. In some cases, theimplement or a portion of the collection implement may remain in thesample tube. A collision between the collection implement and a pipetteor other processing equipment can cause damage, and may result in aspill or other contamination. Also, the sample may include large massesof material, such as large clots or volumes of blood, that may interferewith regular processing operations. Collection implements, large clots,and other foreign objects may appear on the backscatter data assignificant reflections that do not follow the normal patterns of randomscattering from distributed cells or reflection from the liquidmeniscus. In addition, a foreign object is likely to be stationary, asopposed to the moving mass of cells, and its reflected signal shouldlikewise remain stationary. FIG. 12 provides a schematic example ofbackscatter data that might be generated as the signal reflects off acollection implement 810, such as the one shown in FIG. 8. Here, theimplement 810 causes a distinct peak at t_(x) in the reflectionamplitude at a relatively discrete location along the curve, indicatinga possible foreign object.

The backscatter data may be processed in one or more ways to evaluatecell concentration, sample volume, and the presence of foreign objects.It will be appreciated that any number of algorithms or logical processmay be used, ranging from simple point sampling to more complex waveformmorphology interpretation using frequency spectrum analysis and thelike. For example, Fourier analysis may be used to extract frequencyinformation from the raw data to identify time-domain information suchas the time of flight to the last large reflection to indicate theliquid depth. Another process may be to compare successive reflectionpatterns to identify reflections from stationary objects (e.g., a samplebrush) that might be overlaid by reflections from moving cell, andremove such signals to enhance the perception of the reflections fromthe cells. Such algorithms are generally known in the art, and examplesof processing methods are provided in U.S. Pat. Nos. 6,796,195;7,523,649; 7,543,480; 7,739,911; and 7,838,296, which are incorporatedherein by reference. Other variations will be apparent in view of thepresent disclosure. Such analyses can be used to determine numericalproperties (e.g., volume or cell count), or to determine whether thesamples fall without predetermined parameters (e.g., pass/failanalysis). Suitable parameters may be developed using routine empiricaltesting based on control samples, as commonly done in the art.

It has been found in practice, that ultrasonic sample adequacy systemscan successfully identify fluid volume and the presence of foreignobjects, but in some cases the cells themselves may remain essentiallytransparent to the ultrasonic signals. Even in this case the systemstill can provide a significant benefit. If it is necessary to alsodetect cell concentration, measures may be taken to differentiate thecells from their environment, such as by changing the composition of thefluid media, altering the processing temperature, and so on.

As will be apparent from the foregoing, an ultrasonic sample adequacysystem, such as the examples described herein, can provide an advantageby evaluating multiple sample properties with a single, rapid test.Also, unlike nephelometer and turbidity meter equipment, ultrasonictesting does not require an optically clear tube. While the foregoingthree sample properties may be determined essentially simultaneously inone embodiment, other embodiments may evaluate the sample for fewer orother properties. For example, in another embodiment, the ultrasonicsample adequacy system may be used to determine whether a pellet ispresent in a sample tube. In such a system, the backscatter data mightbe evaluated to identify a distinct scattering pattern at a locationbelow the meniscus.

To provide greater versatility, the ultrasonic sample adequacy detectormay be configured to receive tubes having a variety of shapes and sizes.If necessary, adaptors may be included to fit smaller tubes to largerreceptacles. Also, the coupling medium may be generally flat to contactflat-bottomed tubes, but have a dimpled recess to contact tapered tubes.Still further, the detector may have multiple receptacles to facilitatesimultaneous testing of a number of tubes, such as all of the tubes inan entire tube strip 210 or all of the tubes in an entire tube stripholder 212. In one embodiment, a processing module, such as processingmodule 400, may have two sample adequacy test stations: one for testingone kind of tube (e.g., a PreservCyt® tube), and another for testinganother kind of tube (e.g., a SurePath™ secondary conical tube). Theuser may swap out stations depending on the type of tube being tested,or the processing module may direct the tubes to the appropriate teststation automatically based on reading identifying marks on the tubes(e.g., barcodes and associated tube type data) or by followinginstructions provided by a user. Other variations will be apparent topersons of ordinary skill in the art in view of the present disclosure.

Depending on when the sample adequacy test is performed, it may benecessary to resuspend the cells in the sample prior to or duringadequacy testing. For example, in the process described above withreference to FIG. 5, if the sample adequacy is determined shortly afterstep 506 (hydraulically mixing the sample), no further mixing may berequired. In contrast, if the sample adequacy is measured before thetubes 404 are even decapped, such as before the process of FIG. 5begins, mixing may be necessary. Testing prior to decapping may befavorable to forego the need to decap and expose the sample if testingwould not be productive. If mixing is required, a conventional mixer maybe used prior to placing the tube in the adequacy detector.Alternatively, the adequacy detector may be mounted on a platform thatoperates as a mixer, so that the adequacy detector receptacle canoperate as a mixing device to resuspend the cells. Other variations willbe apparent to persons of ordinary skill in the art in view of thepresent disclosure.

Exemplary Decanting Systems

The foregoing examples of processing modules also may include one ormore devices for removing supernatant from a pelletized sample. Such adevice may be configured to match a manual decanting process'smechanics, results, or both. One example of a device for removingsupernatant is a decanting system that rotates the tubes to pour out thesupernatant. A decanting system may decant the individual tubes, butmore preferably simultaneously decants supernatant from all of thesamples in a tube strip 210. An exemplary embodiment of a decantingsystem is now described with reference to the examples illustrated inFIG. 13A-16B.

In general terms, the decanting system 1300 comprises one or moredecanting grippers 1302 that cooperate to rotate a tube strip 210 todecant the supernatant. In the shown exemplary embodiment, the tubestrip 210 is rotated by moving the decanting grippers 1302 verticallyalong a gear rack 1304. It may be desirable to mount the gear rack 1304and/or decanting grippers 1302 on resilient mounts to absorb impact thatmight occur as the decanting grippers 1302 move into engagement with thegear rack 1304. This shock absorption may be particularly helpful wherethe automated drive equipment is sensitive to impacts, or the parts areliable to be damaged or fatigued by repeated impact loads. Any form offlexible mount may be used. For example, the gear rack 1304 may bemounted on a base 1310 by pins 1312, with a slot 1314 surrounding onepin 1312 to allow some rotation about the other pin 1312. A spring 1316is provided to bias the gear rack 1304 into the unflexed position. Asanother alternative, the gear rack 1304 may be cantilever mounted in apocket formed of resilient material that allows some movement betweenthe gear rack 1304 and the platform upon which they are mounted, orother arrangements may be used.

FIG. 13A illustrates the decanting system 1300 in the ready state priorto decanting. FIG. 13B illustrates the decanting system in the decantingposition, in which the decanting grippers 1302 have been moved downalong the gear rack 1304 to rotate the tube strip 210 to allow theliquid contents to flow out by gravity. An opening 1306 leading to adecant waste well 226 is located below the tube strip 210 to receive thedecanted fluids. The decant waste well 226 may include suitablereceptacles, pumps, hoses, or the like to remove and contain thedecanted liquid.

The decanting grippers 1302 may be operated as a separate mechanismwithin the processing module, such as a dedicated robotic arm system.However, in a more preferred embodiment the decanting grippers 1302 areoperated by pipette channels 1308 or other transport mechanisms alreadyin the processing module. In addition to holding the tube strip 210during decanting, the decanting grippers 1302 also may be configured toretrieve the tube strip 210 from a tube strip holder 212 or other holderbefore decanting, and return it to the holder after decanting.

As shown in FIGS. 14 and 15, each decanting gripper 1302 has a base 1400and a rotatable arm 1402. Any suitable arrangement of bearings, axlesand the like may be used to connect each arm 1402 to the respective base1400, to permit relative rotation through a limited or unlimited rangeof motion. One arm 1402 has a gear 1404, which may be a complete gear(i.e., a gear that has teeth around its entire perimeter, to allow full360° motion) or a partial gear (i.e., a set of teeth that does notextend around the entire perimeter, to provide limited rotation). Eacharm 1402 also includes a tube holder 1406 that is adapted to hold thetube strip 210 throughout the operating range of rotation. Forillustration, FIG. 14 shows the tube holder 1406 on the left-handdecanting gripper 1302 in the inverted orientation, and the tube holder1406 on the right-hand decanting gripper 1302 in the uprightorientation.

Each decanting gripper 1302 also may include an attachment interface1408 that can be engaged by a robotic pipettor channel or othermechanism. The attachment interface 1408 may comprise, for example, acup-like receptacle having ribs or grooves on its inner wall that snapinto or are locked into engagement with a pipettor channel's outersurface. Such attachment interfaces are known in the art, and availablecommercially from companies such as Hamilton Robotics of Reno, Nev.(e.g., the Hamilton CO-RE system).

The gear 1404 is selected to engage the gear rack 1304 to rotate thearms 1402 as the decanting grippers 1302 are moved vertically along thegear racks 1304. The shown embodiment uses conventional gear teeth, butthese may be replaced by other kinds of teeth or surfaces. For example,the gear rack 1304 may comprise a simple metal post, and the gear 1404may comprise a rubber traction wheel that can frictionally grip the postto provide the necessary rotational movement. Also, the gear 1404 may bedirectly mounted to the arm 1404 to directly rotate it, as shown in FIG.15, or the gear 1404 may rotate the arm 1404 through one or moreintermediate gears.

The decanting grippers 1302 also may include one or more features toresiliently hold the arm 1402 in one or more positions relative to thebase 1400 when the decanting grippers 1302 are not engaged with the gearrack 1304. This may be desirable, for example, to hold the tube strip210 upright before and after it is decanted, and to prevent the tubestrip 210 from swinging during transport to and from the decantingsystem 1300. In one embodiment, a suitable retaining force may beprovided by simple friction between the arm 1402 and the base 1400, butas the device wears is may be necessary to adjust the friction level tomaintain the desired retention force. In another more preferredembodiment, a resilient holding force may be generated by magnets orother devices.

An example of a magnetic holding system is shown in FIG. 15. In thisexemplary embodiment, the base 1400 may include a magnet 1502 that isretained in a bore 1504 in the base 1400, and the arm 1402 may includeone or more magnets 1506, 1508 that face the base magnet 1502 atrespective predetermined arm orientations. The magnets magneticallyretain the arm 1402 with respect to the base 1400 at the predeterminedarm orientations. The magnets should be oriented to establish thedesired magnetic attraction or repulsion forces to provide the desiredretaining force. The embodiment of FIG. 15 shows the arm 1402 having twomagnets 1506 and 1508, which are located to hold the arm 1402 in theupright and inverted positions by magnetic attraction with the magnet1502 in the base 1400. If desired, one of the magnets may be removed toprovide a magnetic hold in only one orientation (e.g., the uprightorientation), more magnets may be added, the magnets may be replaced bymaterials attracted to magnets, or both magnets may be removed if nomagnetic holding is desired.

The arrangement of magnets in FIG. 15 is exemplary. It will beunderstood that other locations and numbers of magnets may be used inother embodiments. Furthermore, the magnets on either the arm 1402 orthe base 1400 may be replaced by a ferromagnetic material (e.g., iron)or paramagnetic material that is attracted to the magnet(s) on the otherpart. For simplicity, the term “magnet” is intended to cover any kind ofmagnetic material or electromagnet, as well as any material (e.g., aniron slug) that is attracted to magnetic forces. Any combination of theforegoing may be used to provide a magnetic holding force between thearm 1402 and the base 1400.

It has been found that magnets are particularly useful to hold the tubestrip 210 vertically before and after decanting, but allow some minorrotation as the decanting grippers 1302 are moved into initialengagement with the gear racks 1304. This rotation can account for anymismatch between the teeth on the gear 1404 and the rack 1304.Preferably, the magnets permit a mismatch of up to about one half of thegear pitch, so that the gear 1404 can rotate to engage the gear rack1304 even if the teeth contact tip-to-tip as the decanting grippers 1302are moved into initial engagement with the gear rack 1304.

Other embodiments may use other mechanisms to hold or bias the arm 1402into predetermined orientations with respect to the base 1400. Forexample, a return spring may be used to urge the arm 1404 towards theupright position, but allow some rotation to permit initial engagementbetween the gear 1404 and gear rack 1304. Travel stops also may be usedto prevent the arm 1402 from rotating past desired orientations.

FIG. 15 also shows various other exemplary details of a decantinggripper 1302 construction. In this case, the base 1400 has a bore 1510that receives a pair of bushings 1512, and a shaft 1514 is rotatablymounted in the bushings 1512. The gear 1404 and arm 1402 are fixed tothe shaft 1514 by respective pin 1516 that are pressed into respectivebores 1518 in the shaft. The gear 1404 and arm 1402 are spaced from thebase 1400 by washers 1520, to provide a low-friction contact at thislocation.

In use, the decanting grippers 1302 may be stored in a gripper parkinglocation 228 until decanting is required. In preparation for decanting,a decanting grippers 1302 are mounted to respective pipette channels1308 by moving the pipette channels 1308 into the grippers' respectiveattachment interfaces 1408. The pipette channels 1308 may lock into theattachment interfaces 1408 by snap fitment, mechanical locks, magneticlocks, or the like. Next, the pipette channels 1308 are operated tograsp a tube strip 210 with the decanting grippers 1302, and carry thetube strip 210 to a location adjacent the gear rack 1304. The pipettechannels 1308 then move to engage the decanting gripper gear 1404 withthe gear racks 1304. After engagement, pipette channels 1308 are moveddownward to rotate and decant the tube strip 210 via driving engagementbetween the gear 1404 and the gear rack 1304. During decanting, the tubestrip 210 rotates about a decanting axis defined by the rotation centersof the gear 1404. After all the tubes in the tube strip 210 aresufficiently decanted, the pipette channels 1308 move up or down torotate the tube strip 210 to the upright position via driving engagementbetween the gear 1404 and the gear racks 1304. Alternatively, thedecanting grippers 1302 may move out of engagement with the gear rack1304 after decanting is complete, and some other mechanism or motion(e.g., contacting the tubes strips 210 with a fixed object) may be usedto return the tube strip 210 to the upright position.

The operating parameters of the decanting process may be modified toprovide the desired decanting properties or results. Parameters that maybe modified include the rotation rate to invert the tube strip 210, therotation rate to return the tube strip 210 to the upright position, thedecanting angle at which the tube strip 210 is rotated in the invertedposition, and the amount of time spent at the decanting angle. Complexdecanting motions, such as stopping at various angles for various times,may also be used. Other factors that may be considered are thecomposition of the supernatant (e.g., buffers high in alcohol may sheetand not flow rapidly), the pellet properties (e.g., likelihood ofsliding down the tube wall), the composition of the tubes (wetting angleand the like can affect decanting), the diameter and length of thetubes, and so on.

In an embodiment in which the decanting system 1300 is used to simulatemanual decanting in the HC2 protocol, samples in a PreservCyt® buffermay be decanted by rotating the tube strip 210 to an angle of 150° fromvertical, holding for approximately one second (or five seconds or less,in another exemplary embodiment), and returning the tube strip tovertical. To decant samples in a SurePath™ buffer, the tube strip 210may be rotated through 360° with a pause of approximately 0.5 to 1second at 210° from vertical. Other decanting processes may be used tosimulate the performance or results of the HC2 manual decantingprotocol, or to simulate the performance or results of other protocols.

In an alternative embodiment, both decanting grippers 1302 may havegears 1404, two engage two respective gear racks 1304. However, it hasbeen found that embodiments having multiple gear racks 1304, may requireparticular attention to ensure that the gear racks 1304 simultaneouslyengage the gears 1404 on the two decanting grippers 1302. If both gears1404 and both gear racks 1304 have meshing teeth, then any misalignmentpresent at one gear/rack pair that is not also present at the othergear/rack pair can cause a problem with engagement. For example, if onegear 1404 must rotate to properly engage its respective gear rack 1304,but the other gear 1404 is aligned to mesh with its gear rack 1304without rotating, it may be necessary to twist the tube strip toproperly mesh both gears at the same time. In extreme cases, this kindof mismatch may prevent proper tube strip decanting. To prevent thisissue, a one gear rack 1304 may have teeth, and the other gear rack 1304may lack teeth. In this embodiment, only the toothed gear rack 1304 willmesh with its respective gear 1404, and the gear 1404 on the otherdecanting gripper 1302 will simply slide or roll along the toothlessgear rack 1304. In this case, the gear 1404 on the decanting gripper1302 that contacts the toothless gear rack 1304 may be replaced by asimple wheel.

Embodiments using a rack-and-pinion gear system, as described above,have been found to provide simple and accurate decanting, while avoidingthe need for adding expensive additional equipment to the processingmodule. Nevertheless, it will be appreciated that, in other embodiments,any other mechanism may be used to rotate and decant the tube strip 210.For example, an electric motor (e.g., a servo motor) may be mounted onone or both of the decanting grippers 1302 to rotate the tube strip 210.In the shown embodiment, two decanting grippers 1302 are provided, withone decanting gripper 1302 holding each end of the tube strip 210, butit will also be understood that any other suitable arrangement of one ormore decanting grippers 1302 may be used. For example, other embodimentsmay use a single decanting gripper located at the end or somewhere alongthe span of the tube strip 210.

As noted above, the decanting grippers 1302 hold the tube strip 210throughout the range of rotation during the decanting process. FIGS. 16Aand 16B illustrate the decanting grippers 1302 holding a tube strip 210in the upright and inverted positions. The inverted position of FIG. 16Bis fully inverted (i.e., 180° from upright), but the inverted positionused during decanting may instead be at a lesser range of travel (e.g.,160° from upright).

The tube holders 1406 and tube strip 210 may have any suitablearrangement of interacting features to provide the necessary grip toobtain the desired range of travel. In the exemplary embodiment of FIG.17, the tube holders 1406 engage channels 1700 located at each end ofthe tube strip 210. Each channel 1700 may be formed between an upper rib1702 and a lower rib 1704, but other structures may be used to form thechannels 1700. The tube holders 1406 are shaped to fit in the channels1700, and generally conform with the shapes of the channels 1700 toprevent shifting or other unregulated movement as the tube strip 210 isinverted. In this case, the tube holders 1406 have semicircular cutouts1410 that match the semicircular inner wall of each channel 1700, butother shapes may be used in other embodiments.

While it is not required for the tube holders 1406 to hold the ends ofthe tube strip 210, doing so is expected to provide a convenientarrangement in which the decanting grippers 1302 do not interfere withthe tube strip's path of rotation. However, it may not be possible toconveniently access channels 1700 that are located at the ends of thetube strip 210 when the tube strips 210 are placed in an end-to-endarrangement, such as when they are mounted on a tube strip rack 208 withlittle or no gap between adjacent tube strips 210. In this situation, itmay be desirable to provide additional features on the tube strip 210 toallow manipulation if the tube strips 210 when the channels 1700 areblocked. For example, the tube strip 210 may include inboard tabs 1706by which the tube strip 210 may be lifted and manipulated.

FIG. 18 illustrates an example of transport grippers 1800 that may beused to grasp the inboard tabs 1706 to lift and move the tube strip 210.Each transport gripper 1800 includes an attachment interface 1802, suchas described above, that engages with a pipettor channel or othermovable mechanism, and one or more hooks 1804 shaped and sized to wraparound respective inboard tabs 1706. In the shown embodiment, eachtransport gripper 1800 has two hooks 1804 that straddle the top of thetube strip 210 and hold respective inboard tabs 1706 on either side ofthe tube strip 210. As shown in FIG. 19, the hooks 1804 on one transportgripper 1800 may face towards or away from the hooks 1804 on the othertransport gripper 1800, so that the tube 210 cannot slide off the hooks1804 during transport.

The transport gripper 1800 may be stored in a suitable gripper parkinglocation 228 when they are not mounted for use. In other embodiments,the transport grippers 1800 may be replaced by other mechanisms, and maybe provided as a separate robotic mechanism that is dedicated totransporting the tube strips 210.

The exemplary tube strip 210 may be modified in other ways to facilitatetransport and decanting. For example, it will be readily appreciatedthat the foregoing channels 1700 may be placed on the tube holders 1406,and the tube strip 210 may have protrusions that fit into the channels.Similarly, the inboard tabs 1706 may be replaced with cavities intowhich the hooks 1804 fit. The channels 1700, tabs 1706, andcorresponding features also may be replaced by other structures thatfacilitate lifting, moving and rotating the tube strip 210. For example,the tabs 1706 may be omitted and replaced with an attachment interface,such as those described above, that mates to a pipettor channel. Theseand other variations will be apparent to persons of ordinary skill inthe art in view of the present disclosure.

Referring back to FIG. 17, other features of the exemplary tube strip210 are described. The tube strip 210 is used to hold and move thesamples, and may be the primary device for holding the samples generallythroughout a processing module. The tube strips 210 may be made of anymaterial that is suitable for the various processes, which may includetransport, sample adequacy detection, mixing, centrifuging, incubating,decanting, chilling (for storage, if necessary), and other processes. Inan exemplary embodiment, the tube strip 210 may be formed from a naturalpolypropylene homopolymer, which can be shaped to handle all of therequired loads, and is thermally stable. Another suitable material maybe a clarified random polypropylene copolymer, such as 5112C3 availablefrom Pinnacle Polymers of Garyville, La.

The tube strips 210 may have any number or arrangement of individualtubes 1708. In this case, there are four tubes 1708, which convenientlymatches the number of pipettors on a typical robotic four-channelpipettor system having variable spacing between adjacent channels. Suchpipetting systems are available, for example, from Hamilton Robotics orReno, Nev. Each individual tube 1708 may have any suitable shape, suchas a conical bottom joined to a cylindrical sidewall. It will beunderstood that the cylindrical sidewall may not be perfectlycylindrical, and preferably has a slight conical shape (often referredto as a “draft angle”) to facilitate a plastic molding process. Forexample, the cylindrical sidewall may have a 1° conical angle (i.e., theangle of the wall measured relative to the center axis of the tube1708). The tubes 1708 are joined by a common central structure 1710 onwhich the channels 1700 and inboard tabs 1706 are formed. The entiretube strip 210 may be molded as a single part or assembled from variouspieces.

The tubes 1708 may be constructed to help correlate the automatedprocess with existing processing protocols. For example, where theautomated process is intended to correlate to a manual HC2 protocol, thetubes' material and geometry (e.g., diameter and cone angle) may matchtubes used in manual HC2 processing. Matching the material (e.g., usinga natural polypropylene homopolymer) replicates the wetting and contactangle properties of such tubes, which may be helpful to correlate theautomated decanting process with the manual decanting process. Thediameter of the cylinder and shape of the conical bottom also may matchconventional HC2 centrifuging tubes to produce known pellet formationand retention qualities during centrifuging. For example, it has beenfound that using tubes 1708 with the same material and cone geometry aconventional HC2 centrifuge tube provides suitable centrifuging anddecanting results. Some modifications to the conventional tube shape maybe made. For example, the decanting results may be improved bydecreasing the height of the cylindrical sidewalls of the tubes in thetube strip 210, as compared to tubes used in a manual process, to permitfluid to decant more rapidly and reduce the likelihood of slinging fluidout of the tube in an uncontrolled manner. For example, the tubes in thetube strip may have an inner diameter of about 14.7 millimeters, a coneangle of about 30°, and a hemispherical tip having a radius of about 2.2millimeters, which are all similar to conventional HC2 centrifuging tubedimensions, but an overall height of only about 58.2 millimeters, whichis significantly shorter than a conventional HC2 centrifuging tube.

While the illustrated tube strip 210 is constructed with a material andshape to match an existing manual HC2 protocol, this is not necessary inall embodiments of tube strips 210 that are used to perform HC2protocols. Deviations that do not affect the pellet formation resultsmay be implemented as desired. Also, where the process is beingperformed is not a HC2 protocol, other constructions for the tubes 1708may be used to conform with tube structures used in other protocols. Itwill also be appreciated that the shapes of the tubes may not correlatedto other protocols, and may instead be designed to provide the resultsnecessary for the particular application.

The tubes 210 preferably are arranged in a single row that extendslinearly between the channels 1700, so that the tube strip axis (thedirection in which the single row of tubes 1708 extends) is generallyparallel with the decanting axis. The use of a single row of tubes 1708with the tubes 1708 arranged along the decanting axis may be preferredover two-dimensional arrays of tubes or other orientations, because thisarrangement helps reduce the possibility of one tube 1708 pouring intoanother tube 1708 during decanting. The lips 1712 of the tubes 1708preferably are even with one another, and may be located at or near thedecanting axis. Such placement may better simulate a manual decantingmotion and avoid fluid slinging out of the tubes by centripetal forcecaused by the rotation. However, some offset between the decanting axisand the lips 1712 or the centerline of the row of tubes 1708 may beprovided, if desired, to potentially benefit the decanting results.

Exemplary Blotting Systems

After decanting, it may be desirable to blot the tube strip 210 to helpremove supernatant in the tubes, or to remove supernatant that might beclinging to the lips of the tubes. An example of a blotting system 2000is shown in FIGS. 20-27C. The blotting system 2000 generally comprises ablotting sheet supply 2002, a disposal arm 2004, and a waste chute 2006.As shown here, the blotting system 2000 is integral with a decantingstation 222 having a decant waste well 226 and a retainer 224 to hold astrip holder 212. The decanting station 222 also may have a gripperparking station 228 that holds decanting grippers 1302 and transportgrippers 1800 when they are not in use. While these parts areconveniently integrated into a single unit, they may be provided asseparate assemblies or remotely from one another. Where the blottingsystem 2000 is adjacent a decant waste well 226, the blotting system2000 also may include a drip tray 2008 spanning the distance from thedecant waste well 226 to the blotting sheet supply 2002 to catch anydripping liquid and convey such liquid to the decant waste well 226.

In general terms, the blotting system 2000 operates by contacting thelips 1712 of the inverted tube strips 210 with an absorbent material asufficient number of times to remove any liquid clinging to the tubelips 1712. One or more contacts may be sufficient, and each successivecontact preferably is on a clean part of the absorbent material. Thecontact duration may be modified, as necessary to obtain the desiredresults. Longer contact durations also may be used to potentiallyincrease the volume of liquid that is absorbed by virtue of capillaryflow or other fluid mechanics. The blotting parameters (number andduration of blots, orientation of the tubes, etc.) may be selected tosimulate the process and/or results of a manual blotting process, suchas the manual blotting process used in the HC2 protocol. Once blottingis complete for a tube strip 210, the absorbent material is replaced ormoved so that the next tube strip 210 contacts uncontaminated material.Any suitable mechanism may be used to invert and blot the tube strips210. Similarly, and suitable mechanism may be used to provide a cleansupply of absorbent material. The absorbent material may be provided asa roll, individual sheets, or in other suitable forms.

Referring in particular to FIG. 21, an exemplary blotting system 2000may use the previously-described decanting grippers 1302 mounted onrespective pipette channels 1308 to hold and blot the Inverted tubestrip 210. The decanting grippers 1302 may include magnets or otherretainers to hold the tube strip 210 in the desired inverted positionthroughout blotting. Magnets or other resilient holding mechanisms maybe provided in the decanting grippers 1302 to allow some rotation topress the tube lips 1712 flat even on an uneven absorbent materialsurface. It will be appreciated that the orientation for blotting may bedifferent than the orientation used for decanting, and, if this is thecase, the tube strips 210 may be rotated to the desired decantingposition using mechanisms described above or the like. Blotting mayoccur immediately after decanting to avoid keeping the tube strip 210inverted longer any than necessary, and to prevent having to turn thetube strip 210 back to the upright position for storage before blotting.

In this embodiment, the paper supply 2002 comprises a renewable supplyof absorbent sheets 2100 comprising paper or another absorbent material.Suitable absorbent sheets include conventional 4-ply low-lint absorbentpaper towels used in the manual HC2 assay protocol and sheets made ofother paper or nonwoven compositions. The sheet size may be selected toaccommodate the number of blots, tube size, and other factors. Eachsheet 2100 may have a liquid-impervious backing to prevent contaminationof lower sheets, or an impenetrable barrier, such as a removable film,may be provided between sheets in the stack.

The paper supply 2002 may comprise a box-like structure having sidewalls2102, an open top, and a lip 2010 surrounding some or all of the opentop to retain the sheets 2100. As shown in FIG. 20, the lip 2010 mayhave a gap 2012 to provide access for a paper disposal mechanism and tofacilitate paper removal. A movable platform (not shown) is locatedinside the box to hold the sheets 2100, and the platform is biasedupwards towards the open top by one or more springs, air pressure, or asuitable mechanism, to maintain a supply of sheets 2100 at the open topof the paper supply 2002. As this construction is generally in the formof a common spring-loaded paper dispenser, further explanation of theworking parts is not necessary here.

A suitable monitoring system may be provided to detect and report thestatus of the paper supply 2002. For example, a vertical arrangement ofoptical detectors (e.g., infrared emitter/detector pairs or the like)may be provided to examine the paper supply 2002 through one or moreopenings in the sidewalls 2102. Any monitoring technique may be used.For example, the detectors may be used to determine when the sheets areno longer present at the particular level of the respective detector, orthey may register the passing of a reference marker, such as a mark onthe edge of the spring-loaded platform that holds the sheets 2100. Inone exemplary embodiment, four detectors are provided in a verticalstack to evaluate whether the supply is sufficient for a desired numberof samples to be processed, and also to indicate when the paper supplyis nearing exhaustion. These detectors may be integrated into a controlsystem, and monitored to prevent the module from undertaking automatedprocessing until the supply is replenished to a level suitable to lastthroughout the processing run. Other detection systems and algorithmsmay alternatively be used (e.g., a variable resistor other mechanism tomeasure the height of the platform, or a counter to track the number ofsheets used).

The blotting process may be conducted in a manner suitable to removeunwanted liquid clinging to the tubes. The exact requirements for theparticular intended application may be established using empirical orother testing methods. In one embodiment, the blotting process may beconducted to replicate the results of a manual HC2 blotting process. Inthis case, the manual process calls for approximately six blots untilliquid no longer drips from the tube. It has been found that anequivalent automated process may use fewer blots if the tubes areshorter than conventional tubes. For example, the tubes 1708 may havethe same cone dimensions as a standard 10 milliliter Sarstedt tube, buthave a sidewall that reduces the volume to 6 milliliters. In thisconfiguration, it is expected that the liquid more readily flows to thebottom of the tube. For this reason, and possibly others, it has beenfound that four or fewer blots may be necessary. It will be understoodthat the embodiments are not intended to be restricted to any particulartheory of operation.

In one exemplary embodiment, the tube strip 210 is decanted over thedecant waste well 226 for at least one or two seconds, then movedaxially (along the tube strip axis) across the drip tray 2008 and overthe topmost sheet 2100. Liquid may continue dripping from the tubes asthe tube strip 210 moves over the sheet 2100, which may causecross-contamination if the tubes are blotted on that part of the sheet2100. For this reason, before blotting begins, the tube strip 210 ismoved laterally (perpendicular to the tube strip axis) a sufficientdistance to clear the portion of the sheet 2100 that may have becomecontaminated. After this movement, the first blot is performed by usingthe pipettor channels 1308 to move the tube strip 210 into contact withthe sheet 2100. Prior to each subsequent blot (if multiple blots areperformed), the tube strip 210 is lifted and moved laterally asufficient distance to clear any liquid deposited on the sheet 2100during the prior blot. The lateral movement also ensures that no tubecontacts a portion of the sheet 2100 that has passed under or contactedanother tube, to minimize the chance of cross-contamination. Theblotting process is repeated until the tubes are deemed, by results ofempirical testing or by other means, to be clear of unwanted liquid.Afterwards, the tubes strip 210 is turned back to the uprightorientation, by means such as described above, and returned to the tubestrip holder 212.

Variations on the above exemplary blotting process will be readilyapparent to persons of ordinary skill in the art in view of thisdisclosure. For example, the sheets 2100 may be traversed laterallybetween successive blots, instead of moving the tube strip 210.

After blotting, the contaminated sheet 2100 is removed to provide a newsheet 2100 for the next blotting procedure. Vacuum pickups,sheet-feeding rollers, and the like may be used to remove thecontaminated sheets 2100. In the illustrated exemplary embodiment, thesheets may be removed by a disposal arm 2004 that lifts eachcontaminated sheet 2100 from the paper supply 2002, and deposits it inthe waste chute 2006. The disposal arm 2004 generally includes a pinchmechanism to grasp the sheet 2100, and a transport mechanism to conveysthe grasped sheet 2100 to the waste chute 2006.

An example of a disposal arm 2004 is shown in FIGS. 22-25. The disposalarm 2004 includes a rotating arm 2200 that is pivotally mounted to asupport 2202 to rotate about an arm axis 2204. The pinch mechanism islocated on the free end of the arm 2200, and comprises a pair of sheetgrippers 2206 that are mounted on respective shafts 2208. The shafts2208 are rotatably mounted to the arm 2200 on bushings, bearings, or thelike. Each shaft 2208 includes a pinion gear 2210 adapted to rotate theshaft 2208. A central rack gear 2212 is slidably mounted to the arm 2100and located to drive the pinion gears 2210 as it slides. The rack gear2212 is between the two pinion gears 2210, and thus the sheet grippers2206 are counter-rotated by sliding the rack gear 2212 along the arm2100.

The sheet grippers 2206 may comprise any suitable structures forpinching and lifting a sheet 2100. Smooth or toothed rubber rollers,radial pins or fingers, and other structures may be suitable. Devicesthat rely on friction with the paper (e.g., rubber rollers) to initiatethe pinching motion may require periodic servicing to remove paper dustand other contaminants that might reduce the amount of friction.

One preferred embodiment for the sheet grippers 2206 is shown in FIGS.23A and 23B. In this exemplary embodiment, the sheet grippers 2206comprise toothed wheels. The shown wheels are metal, for durability, butplastic or other materials may be used in other embodiments. Thecircular perimeter of each wheel may have teeth to provide mechanicalgrip on the sheets 2100. FIG. 23A shows the wheels in the open positionbefore being operated to grip a sheet 2100, and FIG. 23 shows the wheelsin the closed position in which they grip a sheet 2100 (the sheet 2100is omitted from FIGS. 23A and 23B for purposes of illustrating theparts). The wheels move from the open position to the closed position bycounter-rotating, so that the adjacent portions of the wheels pinch andlift the sheet 2100 from the sheet's starting position. In this view,the wheel on the left is rotated counterclockwise, and the wheel on theright is rotated clockwise; thus, the facing portions of the wheels moveupwards to pinch and lift the sheet 2100.

As shown, each wheel may comprise an outer perimeter 2300 that subtendsan approximately 90° arc. The outer perimeter 2300 may comprise acircular shape with a center point that is located at or near therotation axis of the shafts 2208, but other shapes (e.g., elliptical,cardioid, etc.) and/or an offset rotation axis may instead be used. Theouter perimeter 2300 terminates at a leading edge 2302. As used herein,the leading edge 2302 refers to the end of the outer perimeter 2300 thatis in a leading position when the wheel is rotated from the openposition to the closed position. The leading edge 2302 may be a sharpcorner, but it has been found that forming the leading edge 2302 as arounded corner provides a less aggressive gripping motion that is lesslikely to penetrate the top sheet and pick up multiple sheets 2100, andreleases the sheet 2100 more easily. One or more generally triangular orshark fin-shaped teeth 2304 are provided on the outer perimeter 2300,and the teeth may be inclined towards the leading edge 2302, such asshown. As shown in FIG. 23B, the wheels may overlap slightly when theyare in the closed position. This overlap has been found to assist theprocess of removing the sheets 2100, but it is not required in allembodiments.

Various modifications may be made to the foregoing sheet gripper 2206structures in alternative embodiments. For example, each wheel maycomprise a continuous circular disc that may or may not have teeth, orthe teeth may have different shapes (e.g., square or round).

The sheet grippers 2206 may be actuated by any suitable mechanism.Referring to FIGS. 22, 24A, 24B and 25, the exemplary embodimentincludes a drive wheel 2214 that slides the gear rack 2212 to turn thepinion gears 2210. This motion opens or closes the sheet grippers 2206,depending on the direction in which the gear rack 2212 slides. The drivewheel 2214 is mounted on an axle 2500 (FIG. 25) that rotates on the armaxis 2204. A drive gear 2216 is also mounted on the axle 2500. The drivegear 2216 may comprise any driven structure, such as a motor outputshaft or a driven pulley. In the shown example, the drive gear 2216 is atoothed gear that is driven by a motor 2502 (e.g., a stepper motor) viaa toothed belt 2504 or other intermediate drive devices. Such motors andbelts are conventional and need not be described further. The drivewheel 2214 and the drive gear 2216 are rigidly mounted to the axle 2500,so these three parts rotate together as a single unit. The axle 2500 isrotatably mounted to the support 2202, and the rotating arm 2200 isrotatably mounted on the axle 2500. The foregoing parts all rotatearound the arm axis 2204.

As best shown in FIGS. 24A and 24B, the drive wheel 2214 includes a slot2400 that surrounds a pin 2402 protruding from the side of the gear rack2212. Rotation of the drive wheel 2214 relative to the arm 2200 movesthe slot 2400, and the slot 2400 drives the pin 2402 to thereby slidethe gear rack 2212 along the arm 2200. FIG. 24A shows the drive wheel2214 and the gear rack 2212 in a first position, in which the sheetgrippers 2206 are in the open position as shown in FIG. 23A. FIG. 24Bshows the drive wheel 2214 and the gear rack 2212 in a second position,in which the sheet grippers 2206 are in the closed position as shown inFIG. 23B.

The exemplary drive wheel 2214 also includes a first detent 2404 and asecond detent 2406 located on the outer perimeter of the drive wheel2214. The detents 2404, 2406 are separated by a predetermined angle, asmeasured relative to the arm axis 2204. In this case, the angle isapproximately 90°, but other angles may be used in other embodiments. Aspring-biased pin or ball 2408 is located in the rotating arm 2200. Theball 2408 is positioned to engage the first detent 2404 when the drivewheel 2214 is in the first position (FIG. 24A), and to engage the seconddetent 2406 when the drive wheel 2214 is in the second position (FIG.24B). Engagement between the ball 2408 and either detent 2404, 2406resiliently joins the arm 2200 to the drive wheel 2214, so that thedrive wheel 2214 and arm 2200 rotate in unison. Any suitable ball anddetent mechanism (or other resilient holding mechanism) may be used forthis purpose, and the selection of the same will be within the ordinaryskill in the art in view of the present disclosure.

FIG. 26 illustrates the operation of the disposal arm 2004. In step A,the disposal arm 2004 starts in the first position, with the sheetgrippers 2206 open, and the first detent 2404 engaged with the ball2408. With the assembly in this configuration, the motor 2502 rotatesthe entire disposal arm assembly in a first direction, as shown by arrowA, until the sheet grippers 2206 are located above a sheet 2100. At thispoint, the disposal arm assembly contacts a first travel stop 2600 thatprevents further rotation of the arm 2200. In this orientation, thedisposal arm 2004 is in the pickup position, and ready to pick up asheet 2100.

In step B, the motor 2502 continues to rotate in the first direction tothereby turn the drive wheel 2214, as shown by arrow B. Torque generatedby the motor 2502 overcomes the force holding ball 2408 in the firstdetent 2404 to allow the drive wheel 2214 to rotate independently of thearm 2200. This rotation continues until the assembly is in the secondposition with the sheet grippers 2206 closed and the second detent 2406engaging the ball 2408. During this movement, the sheet grippers 2206grasp the sheet 2100. A limit switch, position indicator, or othermechanism may be provided to indicate when the drive wheel 2214 is inthe desired position, or such information may be obtained by analyzingthe motor by a position sensor (e.g., encoder wheel), by acurrent-detection algorithm that detects increased current as the motorencounters resistance to rotation, or by other suitable mechanism.

In step C, the motor 2502 reverses direction to rotate the entiredisposal arm assembly in the opposite direction, as shown by arrow C.Engagement between the ball 2408 and second detent 2406 causes theassembly to rotate as a unit until the assembly contacts a second travelstop 2602. In the shown embodiment, the second travel stop 2604 islocated to allow the arm assembly to rotate about 180° from the positionshown in steps A and B, but other ranges of travel may be provided. Atthis point, the second travel stop 2602 prevents the arm 2200 fromrotating further in the second direction. In this position, the disposalarm 2004 is in the disposal position, and ready to deposit the sheet2100.

In step D, the motor 2502 continues to rotate in the second direction toturn the drive wheel 2214 as shown by arrow D. The torque of the motor2502 separates the second detent 2406 from the ball 2408 to rotate thedrive wheel 2214 independently of the arm 2200. The drive wheel 2214 isrotated until the assembly is in the first position, with the sheetgrippers 2206 open. The motor 2502 may use any suitable mechanism orcontrol system to stop the rotation when the assembly is in the firstposition. At this point, the sheet 2100 is released and allowed to dropdown the waste chute 2006, and the arm assembly is ready to be movedback to the pickup position shown in step A to remove the next sheet2100.

FIGS. 27A-C illustrate the manner in which the sheet grippers 2206 maygrasp and retain the sheet. When the sheet grippers 2206 move from theopen position (FIG. 27A) to the closed position (FIG. 27B), they pinchand begin to lift the sheet 2100. Rotating the disposal arm backwards(FIG. 27C) pulls the sheet 2100 free of the lip 2010. The gap 2012 inthe lip 2010 may provide access for the sheet grippers 2206, and allowthe sheet 2100 to release more easily.

If desired, sensors may be provided to detect whether the sheet 2100 hasbeen picked up and properly deposited in the waste chute 2006. Such maybe accomplished using conventional optical sensors located where thesheet 2100 is expected to pass, such as at various vertical locationsalong the waste chute 2006. Also, additional mechanisms may be providedto assist with disposing of the sheet 2100. For example, compressed airmay be blown downward to move the sheet 2100, provided sufficientsafeguards are provided to prevent the air from disturbing otherprocesses or contaminating the other samples in the module. As anotherexample, the disposal arm 2004 may be operated through one or moreadditional up and down motions (i.e., moving it back and forth partiallyor completely between the pickup and disposal positions as shown in FIG.26) to force down sheets that are trapped in the waste chute 2006.

It will be appreciated that various modifications may be made to theforegoing embodiment. For example, the illustrated drive wheel 2214 hasa complete circular shape, but it may comprise only a portion of acircle, or be formed as one or more rotating arms. As another example,the shown toothed pinion gears 2210 and rack gear 2212 also may bemodified, such as by using smooth friction rollers, or by usingdifferent articulating mechanisms or linkages. Also, in otherembodiments, the slot-and-pin arrangement may be replaced by otherlinkages, gears or other drive arrangements, and the ball-and-detentsystem may be replaced by other resilient holding mechanisms, such assimple protrusions and notches or magnets. Furthermore, the drive gear2216 may be driven by any suitable alternative mechanism, such as areversible DC or AC motor, a pneumatic or hydraulic cylinder, or thelike. As another example, the drive gear 2216 is shown as a toothed gearthat is driven by a belt 2504, but the drive gear 2216 may comprise alever arm or other mechanism. In yet another alternative embodiment, thedrive wheel 2214 and rotating arm 2200 may be separately operated beseparate motors. Other modifications will be appreciated by persons ofordinary skill in the art in view of the present disclosure.

The operating movement also may be modified in other embodiments. Forexample, other gripping motions may be used, such as by orienting thesheet grippers to grasp the top and bottom surfaces of the sheets,instead of pinching only the top surface. The grippers may also beoperated by linkages to provide an articulated non-circular motion.Also, the rotating arm 2200 may be oriented with its arm axis 2204 abovethe sheet 2100 or at other locations, and the rotating arm 2200 may havea different travel path instead of an arcuate motion as shown herein.

The foregoing blotting system 2000 and embodiments thereof have beenfound suitable for performing blotting processes, and simulating manualblotting protocols. However, it will be understood that blotting is notrequired in all embodiments, in which cases the blotting system 2000 isnot required. For example, it has been found that blotting is notnecessary at all if the decanting process is suitable to decant theliquid without leaving a substantial amount of liquid in the tubes or onthe tube lip, and therefore blotting is not required in all embodimentsthat perform an automated HC2 protocol.

Exemplary Heating Systems

As explained above, a processing module may include one or more heatingblocks, such as heater-shakers, to incubate the contents of the tubestrips 210. While heating systems are well-known, it has been found thatconventional heaters can yield inconsistent heat distribution among thetubes, and cause sample volume loss by evaporation or condensation.These phenomena can impair the reliability of a processing system.

Problems associated with incubation processes are particularly likelywhen samples are processed in conventional microtiter deep-well plates.Such plates typically have 24, 48 or 96 sample wells integrated into asingle plate for simultaneous or essentially simultaneous processing.These plates are often made of molded polypropylene or polystyrene anddimensioned according to Society for Biomolecular Screening (“SBS”)conventions for automated processing equipment. Conventional microtiterplates have several shortcomings when it comes to incubation processes.For example, the plastic material that may be selected for itsnon-reactive properties and low cost is not efficient at conductingheat, and the heat conduction path to the various sample wells candiffer greatly. This can lead to the outer wells being heatedsignificantly more or less rapidly than the inner wells. This problemcan be mitigated, in some cases, by forming spaces between adjacentwells and using a heating block with metal protrusions that fit intothese spaces (so-called “hedgehog” heaters). However, the protrusions onsuch devices can be complex and fragile, and it still may not bepossible to fit the protrusions closely to the standard microtiter platewells to provide efficient heat transfer. Such heating block systemsalso are not expected to operate well when the samples or samplecontainers have relatively large volumes. For example, such protrusionsmay not extend very far along the vertical extent of each well, leadingto relatively little heating at the upper portion of each well. Stillfurther, the protrusions may make it difficult to load the microtiterplate onto the heater, particularly if one were to try to make theprotrusions conform closely to the shapes of the wells.

A further problem with conventional plates is that they typically areenclosed by simple flat polystyrene lids that do not seal the individualwells. Some devices have been provided to seal each individual well,such as elastomer plugs and adhesive-backed plastic or metal foils, butthese have not been found to be amenable to removal by automatedprocessing systems. In such cases, evaporation during incubation can bea significant problem. Finally, such plates comprise a two-dimensionalarray of wells, which cannot be decanted without riskingcross-contamination caused by liquid passing from one well to another.

FIG. 28 illustrates an exemplary sample heating assembly 2800 that isexpected to provide improved incubation performance, such as byproviding consistent heating of all samples, and/or reducing lossesattributable to condensation or evaporation. The heating assembly 2800generally comprises a tube strip holder 212 that holds a number of tubestrips 210 (such as those described previously herein), a heating block2802, and a cover 2804. This embodiment is expected to have particularutility for an automated system performing an equivalent to the manualHC2 protocol, but also may be useful for other incubating processes.

The exemplary tube strip holder 212 conveniently is used throughout theprocessing module, including for general transport and holding, heating,and centrifuging. It will be appreciated, however, that the processingmodule may have multiple different tube strip holders 212 that are usedfor different processing steps. The tube strip holder 212 includes anouter frame 2806 that contains a plurality of tube strip wells 2808. Theouter frame 2806 generally surrounds the tube strip wells 2808 andoptionally may be shaped and sized to match the conventional SBS plategeometry. Features such as gripping surfaces 2810 (e.g., grooves orknurled surfaces) also may be provided to assist with positive grippingand preventing slippage during transport. The tube strip wells 2808 areconfigured to receive one or more tube strips 210. The tube strip wells2808 terminate at an upper lip 2816, which may be positioned tovertically support the tube strips 210 during transport and certainprocessing steps. For example, the tube strips 210 may have lower ribs1704 (FIG. 17) that rest on the lip 2816 to support the tube strips 210,while still providing access to the channels 1700 and inboard tabs 1706.Other arrangements of supporting structures may be used in otherembodiments. For example, the tube strips 210 may be supported bylongitudinal walls 2814 and/or other structures. In the shownembodiment, adjacent tube strip wells 2808 are separated byvertically-extending lateral walls 2812, and longitudinal walls 2814 maybe located between adjacent tubes in each tube strip 210. The tube stripholder 212 preferably is made of magnesium or other materials having ahigh thermal conductivity and relatively high strength-to-weightproperties. A magnesium tube strip holder 212 may be machined, molded,cast, or formed by any variety or combination of processing steps. Othersuitable materials may include aluminum or other metals, plasticmaterials, or combinations of materials.

The heating block 2802 comprises a thermally-conductive material thatmay be mounted on a conventional heater-shaker or other heat source.Aluminum is a preferred material for the heating block 2802 because ithas high thermal conductivity, and can convey heat from a heating deviceto the tube strips with relative speed, and it is relatively light andtherefore can be operated as a shaker (e.g., an orbital shaker) withrelatively little effort. Nevertheless, other materials may be used. Forexample, the heating block 2802 may be formed of steel, and maintainedat an elevated temperature or preheated when it is desired to heat thetube strips 210. The exemplary heating block 2802 includes a pluralityassembled to the heating block 2802, the heating wells 2818 may supportthe bottoms of the tubes 1708 to leave a small gap 3000 between thelower ribs 1704 and the upper lip 2816. In this case, the tube stripholder 212 can move vertically by the distance of the gap 3000 before itcontacts and lifts the tube strips 210 out of contact with the heatingblock 2802. The gap 3000 preferably is equal to or greater than theexpected stacked manufacturing tolerances of the relevant dimensions.

The heating wells 2818 surround a predetermined amount of each tube1708. Preferably, the heating wells 2818 are shaped to closely match theouter surface of each tube 1708, in order to maximize contact betweenthe two and provide the most efficient heat transfer. To providepredictable and uniform heating, the surfaces of the tubes 1708 andheating wells 2818 that are intended to contact one another may be madewith relatively high manufacturing tolerances, or otherwise designed toprovide repeatable contact with one another. In the shown embodiment,the wells 2818 closely fit the conical bottom portion of each tube 1708,and are formed with a conical wall 2826 that diverges at a slightlygreater angle than the outer wall of the tube 1708. With these generallymatching geometries, the tubes 1708 are likely to contact the heatingwells 2818 at or near the lowermost portion of each heating well 2818,and the slightly greater conic angle of the heating wells 2818 ensuresthat the tubes 1708 can easily fall into place without undue dragging onthe heating well 2818 walls. In other embodiments, alternative matchinggeometries may be provided to form a contact area between each tube 1708and each heating well 2818. For example, the heating wells 2818 mayextend above the conical portion of each tube 1708, or, if the tubes1708 are cylindrical, each heating well 2818 may have a flat bottom thatclosely matches the bottom of each tube 1708.

As noted above, the tube strip holder 212 may comprise magnesium oranother material having high thermal conductivity properties, tothermally couple the tube strip holder 212 to the heating block 2802 andturn it into an effective part of the heating assembly 2800. Heat istransferred from the heating block 2802 to the tube strip holder 212primarily by conduction at the locations where the tube strip holder 212rests on the heating block 2802. As shown in FIG. 30, the internal shelf2900 on the tube strip holder 212 may contact a relatively large portionof the heating block side regions 2822 to provide a heat transfersurface. To ensure that these parts are in proper contact, gaps may beprovided at other locations where tube strip holder 212 is in closeproximity to the heating block 2802 to prevent those regions fromabutting one another and preventing contact at of heating wells 2818,each of which preferably receives a single tube 1780, as explainedbelow. The heating block 2802 also may have a central region 2820comprising a surface at a first height, and side regions 2822 comprisingsurfaces that are taller than the central region, the purpose of whichis described below. A bore 2830 may be provided in the heating block2802 for inserting a thermocouple to monitor the heating block'stemperature. The heating block 2802 may be mounted on a conventionalheater, such as an electric heater-shaker, or it may have its owndedicated heating element or elements.

The cover 2804 is configured to cover the tube strips 210. As shown, thecover 2804 may include a plurality of protrusions 2824 that arepositioned to extend into each tubes of each tube strip 210. The cover2804 may be made of clear polystyrene or other materials. Polystyrene ispreferred in one embodiment, because it holds its manufacturingtolerances fairly well, and is hydrophobic. If hydrophilic materials areused in other embodiments, they may optionally be treated withhydrophobic coatings or the like. The cover 2804 may comprise multiplelayers that are spaced apart, but joined at their perimeter or otherlocations, to form an insulating air or gas barrier within the coveritself. The cover 2804 also may be have structures on its upper surfaceto facilitate stacking of other covers 2804, tube strip holders 212, orother parts.

FIGS. 29-31 illustrate the manner in which the exemplary heatingassembly 2800 parts may fit together. FIG. 29 is a lateral cross sectionthrough the centerline of one of the tube strips 210. FIG. 30 is alateral cross section through one of the lateral walls 2812 adjoiningtwo tube strip wells 2808. FIG. 31 is a longitudinal cross sectionthrough all of the tube strips 210.

When the parts are fully-assembled, each tube 1708 is located within arespective heating well 2818, and both the tube strips 210 and the tubestrip holder 212 are supported in the vertical direction by the heatingblock 2802. In the exemplary embodiment, the heating wells 2818 supportthe tubes 1708, and the tube strip holder 212 has an internal shelf 2900that rests on the side regions 2822 of the heating block 2802. When thetube strip holder 212 is raised off the heating block 2802, it may movevertically a short distance before it begins to lift the tube strips210. This helps ensure that the tube strips 210 are in close contactwith the heating block 2802, and not held out of contact with theheating block 2802 by the tube strip holder 212. For example, lower ribs1704 at each end of each tube strip 210 may rest on the upper lip 2816of the tube strip holder 212 when the tube strip holder 212 is notmounted on the heating block 2802. But when the parts are the desiredheat transfer surface. For example, a first gap 3002 may be providedbetween the lateral wall 2812 and the central region 2820 of the heatingblock 2802, and a second gap 3004 may be located between a bottom edgeof the outer frame 2806 and the heating block 2802. These gaps 3002,3004 preferably are equal to or larger than the stacked manufacturingtolerances of the relevant parts.

To increase heat transfer to the individual tubes 1807, the tube stripwells 2808 may closely match the shape of the tube strip 210. Forexample, the gap between the tube strip wells 2808 and tubes 1708 may beonly large enough to ensure consistent loading and unloading of the tubestrips 210. Variations in the gap size may be provided to regulate heattransfer to particular tubes 1708 along each tube strip 210 (e.g., endtubes may have smaller gaps for greater heat transfer). In the exemplaryembodiment, the tube strip wells 2808 comprise a series of conjoinedsemicircular walls that closely surround the tube strip 210 andpartially wrap around the upper portion of each individual tube 1708. Asshown in FIG. 28, the ends 2828 of the tube strip wells 2808 may formportions of the outer frame 2806. In this case, the outer frame 2808 maybe contoured to ensure that the ends 2828 of the tube strip wells 2808have a generally consistent thickness to provide more uniform heating inthis region. As shown in FIG. 29, the longitudinal walls 2814 arelocated between adjacent tubes 1708 to help surround the tubes 1708.FIG. 31 illustrates how the lateral walls 2812 also surround portions ofeach tube 1708.

The outer frame 2806 of the tube strip holder 212 may form an enclosurethat generally surrounds the heating block 2802 and tube strips 210.With this configuration, the outer frame 2806 provides a heat-containingenclosure that holds heated air in proximity to the tubes 1708. Thus,any parts of the tubes 1708 that are not closely surrounded by eitherthe heating wells 2818 or the tube strip wells 2808 are surrounded byheated air captured within the outer frame 2806. This may form abeneficial heat convection path to convey heat from the heating block2802 to the rest of the heating assembly 2800, and help isolate thetubes 1708 from irregular cooling patterns that might be caused by theambient environment (e.g., breezes, nearby heat sources, or the like).

The heating block 2802 and tube strip holder 212 preferably areconfigured to provide consistent heating throughout the upper and lowerportions of the tubes 1708. To this end, the heating block 2802 may beconfigured to contact and conduct heat to the outer perimeter of thetube strip holder 212, because the outer walls have a higher heattransfer rate to the ambient environment and may otherwise remain at alower temperature than the interior portions of the tube strip holder212. For example, as explained above, the heat conduction surface fromthe heating block 2802 to the tube strip holder 212 may be formed as apair of raised side regions 2822 on either side of the heating block2802. In this arrangement, heat transfers by conduction to the outersides of the tube strip holder 212 and then conducts inward through thelateral walls 2812 and longitudinal walls 2814 to heat the Innerportions of the tube strip holder 212. At the same time, ambient airsurrounding the heating assembly 2800 may continuously remove heat fromthe outer portions of the tube strip holder 212. When these heattransfers reach equilibrium, the tube strip holder 212 preferably heatsthe upper portions of all of the tubes 1708 at approximately the samerate.

Other embodiments may use other arrangements of heat conducting surfacesbetween the heating block 2802 and the tube strip holder 212. Forexample, the two raised side regions 2822 may be omitted, and replacedby a cutout on the bottom of the tube strip holder 212 that limitscontact with the heating block 2802 to the outer perimeter of the tubestrip holder 212. As another example, the raised side regions 2822 maybe joined at their ends to form a raised wall that extends continuouslyaround the perimeter of the heating block 2802. This may be beneficialif the heating assembly 2800 has a square or circular shape instead ofthe shown rectangular shape. As still another example, the raised sideregions 2822 may be replaced by a plurality of pedestals or othersurfaces of various shapes and sizes that contact the tube strip holder212 at various locations to distribute heat throughout the tube stripholder 212, while preferably also accounting for expected heat loss tothe ambient air, to thereby provide uniform heating. Other variationsand modifications will be apparent to persons of ordinary skill in theart in view of the present disclosure.

The cover 2804 also may form a functional part of the heating assembly2800. A unitary cover 2804 that extends over all of the tubes 1708 andintervening spaces acts as a heat trap that inhibits heated air fromleaving the proximity of the tubes 1708. To improve this heat trap, thecover 2804 may comprise one or more downward-extending skirts to retainheated air under the cover 2804. For example, the shown embodiment hasan inner skirt 2908 and an outer skirt 2910 that extend downward towardsthe heating block 2802. The skirts 2908, 2910 are located around theperimeter of the cover 2804 to help trap heated air under the cover2804. The skirts 2908, 2910 may extend entirely or only partially aroundthe perimeter of the cover 2804. As shown in FIG. 31, the skirts 2908,2910 optionally may straddle a wall 3100 that extends upward from thetube strip holder 212 to form a labyrinthine path to impede the escapeof heated air. The wall 3100 may extend all the way around the perimeterof the tube strip holder 212, or only portions thereof. In the shownembodiment, the wall 3100 is provided only along the longitudinal endsof the tube strip holder 212. The cover 2804 also may assist withuniform heating by effectively sealing each tube 1708, as well as bypreventing condensation or evaporation, as discussed below.

Various aspects of the foregoing exemplary embodiments and variationsthereof are expected to assist with providing particularly beneficialheating performance. For example, the heating wells 2818 provide closecontact with the bottom of each tube 1708, and thus provide efficientheating of the samples contained therein. This is particularly truewhere the entire volume of the liquid sample 2906 is located within thevertical extent of the heating well 2818, as may be the case whenprocessing a sample for HPV testing according to the HC2 protocol.Another benefit is that the tube strip holder 212 provides an efficientheat conduction path to heat the upper portions of the tubes 1708. In apreferred embodiment used for HPV testing according to the HC2 protocol,the upper portion of each tube 1708 contains only air and possibly traceamounts of liquid. Using normal heating systems, the upper portion ofeach tube 1708 and the air within may be heated only incidentally, ornot at all, which may be intentional to prevent unwanted evaporation.However, the lack of heating may contribute to condensation formingwithin the tube, which causes a different kind of sample loss. Incontrast, the exemplary embodiment uses the tube strip holder 212 toheat the upper portion of each tube 1708 (and thus the air, liquid andwater vapor within) to help prevent the formation of condensation. Otherbenefits may be realized and obtained in these and other embodiments.

FIG. 32 is a temperature (“T”) versus time (“t”) plot comparing theperformance of an exemplary heating system as described above with aconventional water bath heating system used in the manual HC2 protocol.A solid line 3200 illustrates the temperature of a sample processedaccording to the manual HC2 protocol. In the manual HC2 protocol, thesample is placed in a 65° (±2°) Celsius water bath 15 (±2) minutes,removed from the water bath and vortexed for 15-30 seconds, and replacedin the 65° (±20) Celsius water bath for 30 (±3) minutes. Line 3200 showsthat the sample temperature raises relatively quickly when it is placedin the water bath, but the temperature experiences a significant drop3204 during the vortexing step. A dashed line 3202 illustrates thetemperature profile of a sample heated using a heating assembly 2800such as described above. The sample temperature rises marginally moreslowly than the sample processed in a water bath, but the temperaturedoes not drop during the vortexing step because vortexing is performedwithout removing the samples from the heating assembly 2800 by mountingthe heating assembly 2800 on a conventional electric heater-shaker. Theresulting time at temperature is similar in both scenarios. As a result,the total incubation time and final results for both samples areapproximately the same.

As noted above, the cover 2804 may be configured to help reduceundesirable sample evaporation and condensation. This benefit may beparticularly desirable when the incubation temperature is high, theincubation time is long, the sample volume is relatively small, or thetube volume is relatively large. For example, in the case of anautomated equivalent to the manual HC2 protocol, all of these conditionsmay exist. In this case, the incubation time may be at 65° Celsius for45 minutes, the sample volume may be approximately 200 microliters, andthe tubes may have a diameter of approximately 14 millimeters. For thisprotocol, it may be desirable to limit losses due to evaporation andcondensation to 30 microliters or less.

FIG. 33 is a cross section of an exemplary cover 2804, shown installedon a tube 1708 mounted in a tube strip holder 212. The cover 2804 mayinclude features intended to limit condensation and evaporation duringincubation. The shape of the cover 2804 is intended for an automatedequivalent to the manual HC2 protocol, but it may be used in otherapplications with or without modification. The cover 2804 comprises aplurality of protrusions 2824 that each fit into a respective tube 1708.Each protrusion 2824 preferably includes an upper seal 3300, a lowerseal 3302, and a lower conic section 3304, as described below.

The upper seal 3300 is formed as a first conical wall that seats againstthe upper lip 1712 of the tube 1708. The first conical wall starts at afirst diameter that is larger than the diameter of the upper lip 1712,and tapers in the downward direction to a second diameter that issmaller than the diameter of the upper lip 1712. The angle of the firstconical wall may be in the range of approximately 15° to 20°, andpreferably is approximately 15°, but other angles may be used asdesired. Direct contact between the first conical wall and the upper lip1712 seals the tube 1708.

The lower seal 3302 is located below the upper seal 3300, and is formedas a second conical wall that approximately matches the draft angle(e.g., approximately 1°) of the adjacent inner wall of the tube 1708.Thus, the second conical wall is approximately parallel to the tube1708. The second conical wall has a diameter that is slightly less thanthe Inner diameter of the adjacent tube wall, in order to create acapillary seal upon contact with liquid that remains at or near thetube's lip 1712 after the decanting process. For example, a gap ofapproximately 0.2 mm to 0.3 mm may be used to form a capillary sealusing the supernatant liquid in the HC2 protocol. Where more or lessviscous fluids are in the tube 1708, the gap may be adjusted accordinglyto provide the desired capillary seal.

The vertical height of the lower seal 3302 may be selected to enhancethe likelihood that a complete capillary seal will be formed, and ataller wall may be likely to contact more residual fluid and thus bemore likely to form a complete capillary seal around the entire wall. Ataller seal also helps isolate the tube contents, even without acapillary seal. However, a taller seal may seal too tightly for reliableautomated processing, and therefore the wall should not be so long as tointerfere with the automated process. In a preferred embodiment, thesecond conical wall that forms the second seal 3302 has a verticallength of approximately 2.5 mm to 3.0 mm.

Is has also been found that the lower seal 3302 may help resist orprevent the cover 2804 from shaking off during mixing operations. Aconical protrusion into a tube 1708 is likely to climb upwards duringrapid shaking, and particularly orbital shaking. The parallel wall ofthe lower seal 3302 does not exhibit this phenomenon, and thereforehelps to hold the cover 2804 in place during mixing.

The use of the foregoing two seals is expected to provide moreconsistent sealing results for all of the tubes 1708 held in a singletube strip holder 212. While the upper seal 3300 may effectively sealthe tube 1708, variations in the flatness of the cover 2804 may cause agap between some of the upper seals 3300 and their respective tubes1708. In those cases, the lower seal 3302 provides a capillary seal thathelps isolate the contents of the tubes 1708 from the ambientenvironment. The foregoing seals also do not require supplementalsealing devices, such as O-rings or the like, and are readily formedusing conventional processing techniques.

It will be appreciated that the foregoing upper and lower seals 3300,3302 may be modified in various ways. For example, the upper seal 3300may be made of a wall that is not conical (e.g., a series of steppedsquare ridges or a curves wall). As another example, there may be a gapbetween the upper seal 3300 and the lower seal 3302. Other variationsand modifications will be apparent to persons of ordinary skill in theart in view of the present disclosure.

The cover 2804 also may include additional features to help seal thetubes 1708. For example, each protrusion 2824 may be surrounded by anouter ring 3306 that extends downward from the cover 2804. The outerring 3306 preferably extends below the upper lip 1712 of the tube 1708to provide a tortuous path between the interior of the tube 1708 and theambient environment. Thus, the outer ring 3306 helps isolate thecontents of the tubes 1708 if the upper and lower seals 3300, 3302 donot form complete seals.

The lower conic section 3304 extends below the lower seal 3302 and intothe tube 1708. The illustrated exemplary lower conic section 3304 hastwo regions having different conical taper angles, and a rounded tip,but these particular features are not required in all embodiments, andthe lower conic section 3304 may be modified or omitted in otherembodiments. As shown in FIG. 33, the lower conic section 3304 also mayextend below the top of the tube strip holder 212. The lower conicsection 3304 may help suppress the formation of condensation as it isheated by the tube strip holder 212, by providing a heated mass withinthe tube 1708 and displacing some volume of air in the tube 1708. It hasbeen found that a protrusion 2824 into the tube 1708 transfers more heatto the tube's contents than a flat cover. It may be desirable to locatethe tube strip holder 212 close to the bottom of the cover 1804 toenhance heat transfer to the lower conic section 3304. Such closeplacement also may help provide a mass of warmed air surrounding thetubes 1708 to prevent local cool spots at which condensation might form.

It has been found that a cover 2804 constructed substantially asdescribed above and shown in FIG. 33 helps reduce evaporation andcondensation. Testing indicates that a relatively minor amount ofcondensation forms on the lower conic section 3304 and inner wall of thetube 1708. The condensation forms at a slightly cooler region locatedabove the level of the tube strip holder 212, and primarily on the lowerconic section 3304 of the protrusion 2824. In these tests, the amount ofliquid lost to condensation and evaporation in the tube 1708 did notexceed the maximum limit for the particular HC2 test protocol. It isexpected that even lower condensation may be obtained by more directlyheating the lid 2804 and protrusions 2824. For example, the lid 2804 andprotrusions 2824 may be heated by a radiant heater or convection heater,or by an upper heating block that contacts the cover 2804 (e.g. heatedprongs that extend down into the protrusions 2824). Other variations andmodifications will be apparent to persons of ordinary skill in the artin view of the present disclosure.

The foregoing arrangement is expected to reduce or minimize air exchangebetween the interior of the tube, with its saturated water vapor, andthe ambient environment. In alternative embodiments, the features of thecover 2804 may be used separately or in combination with otherstructures. Also, the cover 2804 may be divided into separate units,such as units that cover individual tubes 1708 or tube strips 210. Thecover 2804 preferably is formed as a stackable shape, and may includefeatures (e.g., tabs, grooves, opposed surfaces or the like) to permitrobotic manipulation using mechanical grippers, vacuum handlers, or thelike.

It will be appreciated that the various features of a heating system2800 that are described herein may be used collectively, separately, orin various combinations. For example a tube strip holder 212 having heatconducting features may be used in conjunction with a heating block2802, but without a cover 2804. As another example, a cover 2804 havingsome or all of the features described herein may be used withconventional deep well plates or other heating systems. In additionvarious additions may be made to the foregoing embodiments. For example,the heating wells 2802 may include a water supply to enhance heattransfer to the tubes 1708.

Exemplary Tube Strip Holder Features

As noted above, the tube strip holder 212 may be used for a variety ofoperations in addition to heating. As explained previously, the tubestrip holder 212 may be used to hold the tube strips 210 fortransportation throughout a processing module, and may be configured tofacilitate decanting and blotting operations by permitting access bydecanting grippers and the like. Where the tube strip holder 212 is usedin a processing module that performs an automated equivalent of a manualHC2 protocol, it also may be desirable to use the tube strip holder 212during machine vision inspection of the tube contents or duringcentrifuging operations.

Referring to FIGS. 34 and 35, the tube strip holder 212 may be used asan optical mask for a machine vision inspection, such as may beperformed by a vision inspection station 218. During vision inspection,a light source is activated to illuminate the contents of the tubes andone or more cameras or other sensors are used to evaluate the lightpattern to determine whether the tubes contain pelletized samples.Typical optical analysis equipment for conventional multi-well platesuse a light source such as an array of light-emitting diodes passingthrough a diffuser, or other diffuse sources intended to provide uniformillumination to all of the wells. It has been found, however, thatoptical testing of an array of tube strips 210 held by a tube stripholder 212 can cause problems unique to this arrangement. In particular,light passing through the tube strip holder 212, but not through thetube strips 210, can overexpose the sensor, or provide properly-exposedimages of the tubes in the center of the tube strip holder 212 butunderexposed images of the tubes around the perimeter. It has also beenfound that removing one or more tube strips 210 from the tube stripholder 212 causes even more severe overexposure, and may allow light toreflect off the tubes adjacent the missing tube strips 210, causingglare that inhibits proper optical testing.

FIG. 34 illustrates an example of a tube strip holder 212 thatexperiences significant exposure problems during optical testing. Thistube strip holder 212 is formed as a simple rectangle having onelongitudinal cross-wall 3408 and two lateral cross-walls 3410. Thedesign of this tube strip holder 212 is intended to minimize weight (andthus centrifuging loads), and have low material and manufacturing costs.Despite these benefits, the tube strip holder of FIG. 34 has been foundto have problems during optical testing. For example, there is anoverexposed region 3200 where tube strips 210 have been removed, glare3202 caused by the large overexposed region 3200, smaller overexposedregions 3404 between adjacent tube strips 210, and underexposed tubeimages 3406 at the perimeter of the tube strip holder 212.

It has been found that these problems may be overcome by forming thetube strip holder 212 as an optical mask that substantially blocks lightfrom bypassing the tubes, and reduces or prevents glare from reflectingoff tubes when a tube strip 210 is removed. As shown in FIG. 35, thetube strip holder 212 is formed with lateral walls 2812 and longitudinalwalls 2814 that form a separate opening 3500 for each tube. Duringexposure, these walls 2812, 2814 form an optical mask to block a largeportion of the light that would otherwise bypass the tubes and directlystrike the detector, thus reducing the possibility of overexposure. Thewalls 2812, 2814 also isolate the individual tubes openings 3500 toinhibit glare that might otherwise be present when the optical analysisis performed with one more tube strips missing from the tube stripholder 212.

As shown in FIG. 35, the tube strip holder 212 also may includeadditional openings 3502 located between the individual tube openings3500 and around the outer perimeter. Such openings 3502 may be providedto reduce the weight of the tube strip holder 212, and preferably arenot large enough to affect the optical testing results. Such openings3502 also may serve other purposes. For example, one or more openingsmay comprise a registration opening 3504 (e.g., an asymmetric or uniqueshape or pattern of shapes) that indicates the orientation of the tubestrip holder 212. This feature may be particularly helpful when theimage is analyzed during later automated or manual processing steps toquickly and accurately determine the identity of each individual sample.

Referring now to FIG. 36, the tube strip holder 212 may also beconfigured for use in a centrifuge. Centrifuging can generate tremendousloads. For example, a 300 gram object centrifuged at 2,900 gravitiesgenerates a load of 870 kilograms. A tube strip holder weighing 130grams would effectively weigh 377 kilograms, and a loaded tube stripweighing 25 grams would effectively weigh 72.5 kilograms. The effectivemass can be reduced by minimizing the weights of the tube strip holder212, tube strips 210 and samples, but the tube strip holder 212 and tubestrip 210 must be strong enough to remain intact throughoutcentrifuging.

The forces generated by the centrifuge are oriented in a radialdirection from the spin center 3600 to the center of mass of eachcentrifuged object, and masses that are further from the spin center3600 will experience proportionately larger loads. In the case of a tubestrip holder 212 loaded with tube strips, the tube strip holder 212preferably is mounted with the center of the base perpendicular to thespin center, and the mass of the tube strip holder 212 and its contentspositioned between the center of the base and the spin center, such asshown in FIG. 36. This arrangement prevents the creation of unbalancedcentrifuge forces. However, this arrangement does lead to the generationof tangential forces (forces tangential to the rotation path) on theparts. For example, the centrifuge forces 3602 on the tubes and tubestrips 3604 that are offset in the tangential direction from the centerof the tube strip holder 212 will include a tangential component thatwill be exerted against the side of the tube strip holder 212. Similartangential forces would be present at other locations, such as thelateral walls 2812 and longitudinal walls 2814. As will be understoodfrom a simple vector analysis, the magnitude of these tangential forcesincreases as the spin radius decreases.

The tube strip holder 212 preferably is designed to withstand loads ashigh as 2,900 gravities for 15 minutes or more, in a centrifuge having arelatively small spin radius (e.g., less than about 200 mm). To thisend, the tube strip holder 212 may comprise magnesium or other materialhaving a relatively high strength-to-weight ratio. The tube strip holder212 may be constructed to carry all of the forces generated by the tubestrips 210 and samples therein, but in a more preferred embodiment, thetube strip holder 212 cooperates with the centrifuge bucket 3606 todistribute and collectively carry the centrifuge loads generated by thetube strips 210. For example, in the embodiment of FIG. 36, thecentrifuge bucket 3606 includes a base 3608 in which a plurality ofrecesses 3610 are formed. Each recess 3610 receives the bottom of arespective tube 1708, such as shown in FIG. 36. The recesses 3610 arepositioned to hold the tube strips 210 out of vertical supportingcontact with the tube strip holder 212 (such as described above withreference to the heating block). In doing so, the centrifuge 3606directly bears the tube strip's centrifuge loads in the verticaldirection (i.e., the vertical direction of the tube strips 210 and tubestrip holder 212). The bucket 3606 also may directly carry a portion ofthe tangential loads, such as those discussed above, by interactionbetween the recesses 3610 and the bottom tip of each tube 1708. Theremaining tangential loads will be directly applied to the tube stripholder 212, and particularly to the lateral walls 2812. As such, thetube strip holder 212 preferably has sufficient strength to bear suchloads. In the illustrated embodiment, the tube strip holder 212 ismounted in the centrifuge bucket 3606 with its long axis oriented in thetangential direction. As such, the lateral walls 2812 and end walls ofthe tube strip holder 212 should be strong enough to bear the tangentialcentrifuge loads. In this arrangement, using the centrifuge bucket 3606to directly carry a large portion of the centrifuging loads allows thetube strip holder 212 to be lighter and potentially less complicated andexpensive to manufacture.

In alternative embodiments, different load-bearing arrangements may beused. For example, the tube strips 210 may be mounted to swing withintheir respective tube strip wells 2808 to align with the direction ofthe centrifugal forces, which may alleviate the need for the tube stripholder 212 to bear tangential forces.

As will be appreciated from the foregoing description, the exemplarytube strip holder 212 may be constructed to provide a number offunctional features and conveniences that are not found in conventionalsample plates. The tube strip holder 212 holds a number of tubes inremovable strips, and does so in a manner that allows automated removaland replacement of the tube strips 210 for processes such as decantingand blotting. To this end, the tube strips 210 may be provided as singlerows of tubes 1708, which helps prevent cross-contamination duringdecanting, blotting, and other processes. The tube strip holder 212 alsois configured to thermally couple to a heating block to distribute heatto the upper portions of the tubes 1708 during incubation, to helpprevent condensation and ensure even heating of all of the tubes 1708.The tube strip holder 212 is also designed to form an optical mask toimprove the vision inspection process, and to cooperate with thecentrifuge bucket 3606 to contain the tubes 1708 during high-gravitycentrifuging operations. In addition to all of these features, the tubestrip holder 212 may be formed as a reusable part, to thereby saveprocessing expenses.

The combination of the foregoing features in a single tube strip holder212 is particularly beneficial to simplify the operation of automatedprocessing modules, because it may minimize the need to transfer thesamples from one holding device to another during processing. It will beappreciated, however, that it is not necessary in all embodiments toinclude or combine all of the foregoing features into a single tubestrip holder 212, and the various features of the tube strip holder 212may find separate utility in other applications.

Exemplary Vision Inspection Systems

Referring now to FIGS. 37 and 38, examples of exemplary visioninspection systems for detecting the presence of a pellet in a tube aredescribed.

FIG. 37 shows a vision inspection system 3700 having a vision inspectionstation 218 and a camera enclosure 220. The vision inspection station218 includes a platform 3702 configured to hold a tube strip holder 212and its associated tube strips 210. The platform 3702 includes atransparent (e.g., glass, plastic or open) window 3704 through which thebottom of the tube trips 210 are visible. The window 3704 may besurrounded by a groove 3706 in which a lower lip of the tube stripholder 212 rests to ensure proper positioning. If it is determined thatthe inner lip of the groove 3706 blocks the view through the window 3704to the tube strip holder 212, the tube strip holder 212 may be heldwithin a retaining wall to allow a greater view of the parts. Forexample, the window 3704 may be surrounded by a beveled wall into whichthe tube strip holder 212 can be lowered to hold only the outer edge ofthe tube strip holder 212 and allow full viewing of the inner regions ofthe tube strip holder 212. This may be desirable when the tube stripholder includes features such as a registration opening 3504 near theouter perimeter.

The vision inspection station 218 is covered by an upper panel 3708 thatcontains one or more light-emitting diodes (“LEDs”) or other lightsource 3710. The light source 3710 preferably is distributed over anarea and includes a diffuser or other means to distribute the lightsource. In a preferred embodiment, the light source 3710 is a diffuselight that is distributed over an area larger than the area of tubestrip holder 212, to minimize light falloff or dark areas around theedges of the tube strip holder 212. The light source 3710 may emit lighthaving any monochromatic color or polychromatic color range, such as awarm white color.

A mirror 3712 is mounted below the window 3704 to reflect light from thelight source 3710 that passes through the tube strips 210 and tube stripholder 212. The mirror preferably comprises a first surface mirror inwhich the front surface is the reflector, instead of the back surface.This prevents the creation, as typically happens in back surfacemirrors, of a separate “ghost” image reflecting off the front surface.The mirror 3712 preferably is sized to reflect the entire image of thetube strip holder 212 and its tube strips 210 to allow simultaneousinspection of all of the tubes. A camera 3714 is located to view thelight reflected by the mirror 3712. The camera 3714 may comprise anysuitable visual inspection camera, such as a 5 megapixel color detectorcoupled to a 23 millimeter high-resolution lens. In the exemplaryembodiment, the mirror 3712 is inclined at 45° angle to redirect thevertical light along a horizontal path. This provides a convenientconfiguration for the various parts, but other angles may be used inother embodiments. Also, if the camera 3714 is mounted with a directview of the tube strips 210 (e.g., directly below the window 3704 andpointing up), the mirror 3712 may be omitted.

The parts of the vision inspection system 3700 are generally enclosed ina housing that blocks in the ingress of ambient light that mightotherwise affect the test results. The tube strip holder 212 is placedin the vision inspection station 218 through an opening 3716, and thecover 3708 has an overhang 3718 that helps shade the interior of thehousing. If desired, a closable door or other access port may beprovided to more fully block ambient light.

The light source generates a silhouette image of the tube strips 210 andtube strip holder 212, such as the Inspection image shown in FIG. 38. Asexplained above with reference to FIG. 35, the tube strip holder 212 mayform a generally opaque light mask, which creates a large black ornearly black masked area 3802 in the inspection image 3800. The tubes1708 of each tube strip 210 generate somewhat shaded circular tubeimages 3804. The sample pellets generate dark pellet images 3806. TheInspection image 3800 also may include a number of bright through-holeimages 3812, where light passes through through-holes formed in the tubestrip holder 212.

The inspection image 3800 of this exemplary embodiment also exhibits twoother notable features. First, it has been found that empty tubes 1708generate empty tube images 3808 having a dark “sphere-cone” ring 3810caused by light bending through the transition between the conical lowerwall and hemispherical tip of the tube 1708. Other tube shapes may notexhibit this property.

In addition, the camera's location and lens type can result in aparallax effect. Parallax is an angular variation in the line of sightthat depends on the distance to the viewed objects. Parallax causesequally-spaced objects to appear further apart when they are closer tothe viewer, and closer together when they are further from the viewer(i.e., perspective). In the case of the tubes 1708 mounted in a tubestrip holder 212 and viewed from the bottom, the bottom ends of thetubes 1708 (where the sample pellets are likely to be located) willappear on a slightly larger grid pattern than the openings through whichthe upper parts of the tubes pass. This makes the outer rows of tubesappear to spread outward. As shown in FIG. 38, this causes the pelletimages 3806 to appear be displaced from the centers of their respectivetube images 3804. The offset is greater for tubes that are further fromthe viewing axis of the camera 3714, which, in this case, is pointeddirectly at the geometric center of the tube strip holder 212. Suchparallax can be mitigated by moving the camera further from the image,or using a telecentric lens.

The vision inspection system 3700 evaluates the inspection image 3800 todetermine whether each tube includes a respective sample pellet. Thisanalysis may be performed on samples with or without the presence of asupernatant liquid (e.g., before or after decanting). In the context ofa HC2 process, the samples may comprise a transport stabilization mediumliquid containing a small percentage of cellular, blood, mucous and/orother materials obtained with a cervical sample. The sample iscentrifuged to compress the denser, and in this case more opaque,material into a pellet form. In other contexts and embodiments, the tubemay contain other kinds of samples.

In some cases, a sample tube may not appear to include a sample pellet,either because the sample simply was not taken, or because the samplewas not adequate to form a pellet. In these cases, the contents of thetube may still be processed to determine whether they include HPV orother condition. If the results come back positive (i.e., HPV or anothercondition is detected in the sample), then the test results may besufficient despite the lack of a visible pellet. However, if the resultsof the assay are negative (i.e., no HPV or other condition is detected),then the test will be ruled indeterminate because it cannot bedetermined whether it was due to a lack of the condition, or theinsufficiency of the sample. In these cases, a supplemental test may beperformed or another effort may be made to obtain an adequate samplefrom the patient.

Any suitable algorithm may be used to determine the presence of a samplepellet in each tube. In one example, described in detail below, theimage processing algorithm performs two main steps. First, it analyzesthe inspection image 3800 to locate the tubes within the image andcreate respective regions of interest. Second, the algorithm separatelyanalyzes each region of interest to determine whether it contains apellet image. In the following example, there are 24 tubes and 24associated regions of interest, but other quantities may be usedinstead.

The first step of the exemplary process-identifying regions of interest(hereinafter, the “ROI process”)—is detailed in FIG. 39. In generalterms, the purpose of the ROI process is to identify and isolate thetwenty-four tube locations in the image, and remove the datarepresenting the dark masked area 3802 and the bright through-holeimages 3812.

The ROI process begins at step 3900 when the camera 3714 takes theinspection image 3800 of the tube strip frame, tubes and samples. Thesignal gain (light “amplification”) of the camera 3714 may be set at apredetermined level based on an initialization process, or activelycontrolled based on other criteria or feedback, in order to providecontrast levels that are expected to facilitate the remaining processingsteps. If the camera 3714 uses a color sensor (i.e., one that detectsseparate red, green and blue channels or equivalents thereof), thesignal may be processed to extract the red image data as intensityvalues and discard the remaining data (green and blue) for some or allof the remaining process steps. By separating out only one colorchannel, the image data becomes a monochromatic image with intensitybeing measured on a conventional 0-255 scale, with 0 being no intensity(black), and 255 being the maximum intensity (pure red). Intermediatevalues represent different intensities of the light (different darkershades of red). The red channel is selected in this example because itprovides the greatest contrast range for the particular subject matterof the HC2 protocol being performed. The HC2 protocol uses a SampleConversion Buffer that makes the liquid clear red, and the pellet abrownish-red, and thus the variations in the contrast are most visiblein the red color range (blue and green channels would make the contentsdarker and more confusing to distinguish details). Other processes mayuse other color channels, composite colors, greyscale data, or the like.

Once the red channel is extracted, the ROI process may use one or moresearch techniques to search for regions of interest. In this example,three techniques are performed sequentially or in parallel.

The first search technique begins at step 3904. In this step, the ROIprocess filters the image data to find all data points having anintensity of 253 to 255 on the 0-255 scale. This step identifies “holes”where the light passes through the tubes and through-holes withoutlosing much intensity. It will be appreciated that the data points thatform the image each may represent the image data detected by a singlepoint detector (sometimes called a “pixel”) on the camera's imagesensor. However other groupings may be used to identify discrete datapoints (e.g., each point may be the average value detected by multipleadjacent point detectors), and filtering algorithms such as conventionalsharpening masks may be applied before or during the ROI process.

In step 3906, the ROI process “fills” the holes found in step 3904 togenerate solid regions. In step 3908, the ROI process searches the solidregions to find circular areas having a radius between 145 and 200pixels. These circular areas are identified as regions of interest, andareas smaller than this are removed from further consideration. The145-200 pixel range is selected in this embodiment because it is largerthan the sizes of the through-hole images 3812, but smaller than thetube images 3804. Thus, in effect, step 3908 removes the through-holesimages 3812 from further consideration. In other embodiments havingdifferent pixel scales, other dimensions may be used.

In step 3910, the ROI process matches the regions of interest to adefault grid of twenty-four predetermined locations. For example, theROI process may find the center of each circular region of interest, andcompares these centers to points or areas on the grid. This preventsbadly misaligned tube strip holders from being processed incorrectly.Minor deviations may be processed as normal, and larger deviations maygenerate an error signal indicating the need to realign the tube stripholder. It also may be possible to correct deviations by orientingand/or scaling the image to position the regions of interest to matchthe default grid.

Next, in step 3912, the ROI process crops the radius of each region ofinterest by 25 pixels to remove any dark shadows caused by the maskedarea 2809 that might appear in the regions of interest. This is mostimportant in the outer rows of tubes (those furthest from the viewingaxis) because the walls of the tube frame holder 212 appear to overlapportions of the tubes due to the parallax view, and cast shadows overthe outer edges of the tubes. Again, the 25 pixel value is based on thepixel scale of the particular embodiment, and other values may be usedin other embodiments.

In the final step 3914 of the first search technique, the ROI processdetermines whether a region of interest has been identified for eachtube well. If all twenty-four tube wells are imaged, then the processcontinues to step 3944, which is described below. If the tube welllocations are not all found, then processing continues to the secondsearch technique, which begins at step 3916.

Step 3916 begins with the monochromatic image generated in step 3902,and uses an automatic threshold evaluation routine to find regions ofinterest for any of the remaining tube-wells. This process may beperformed on the entire image, or just in particular areas correspondingto tube well locations where no region of interest was found using thefirst search technique. The automatic threshold evaluation routine 3916uses an entropy method or other automatic method to identify and extractmore intense regions from an image containing a mixture of more and lessintense (i.e., lighter and darker) regions. The starting parameters (ifany) and exact entropy model may be selected according to well-knownimage processing techniques, and such methods and the details of suchalgorithms need not be described here.

After step 3916 extracts the more intense regions, the second searchtechnique applies a filter in step 3918 to remove extracted intenseregions having an area of less than 2000 pixels. This filter removesimage “static” and any small specular reflections or through-hole images3812 from further consideration.

The remaining areas identified in step 3916 are then “filled” in step3920 to generate solid regions, and filtered in step 3922 to identifycircular regions of interest comprising circular areas having a radiusbetween 160 and 200 pixels, and remove any other areas from furtherconsideration. In step 3924, the centers of the circular regions ofinterest are matched to the default grid, as described above in step3910. Next, in step 3926, the regions of interest are cropped by 25pixels, as described above in step 3912.

At the end of the second search technique, the ROI process determineswhether a region of interest has now been identified for each of theexpected tube well locations. If so, the process continues at step 3944,described below. If not, the ROI process continues to a third searchtechnique starting at step 3930.

The third search technique comprises a series of steps, starting at step3930 and concluding at step 3940, that generally match steps 3904through 3914 described above. The only difference is that the thirdsearch begins in step 3930 by filtering the image data to find all datapoints having an intensity of 85 to 255 on the 0-255 scale. Thus, thethird search technique uses a lower intensity cutoff threshold toidentify regions of interest. This technique is expected to identifytube images 3804 that are darker and have less contrast than thoseidentified by the first two techniques. At the end of the third searchtechnique, the ROI process once again determines whether a region ofinterest has been identified for each expected tube well location. Ifso, the process continues to step 3944. If not, the ROI processcontinues to step 3942, where it generates an error signal indicatingthat the ROI process was unable to identify a region of interest foreach tube well location. In this case, the entire vision inspectionprocess may cease for the particular tube strip holder, or it maycontinue for any tube well locations that are successfully identified bya region of interest.

FIG. 40 is an exemplary output image generated by performing the imageprocessing algorithm on the inspection image 3800 of FIG. 38. Thetwenty-four circles indicate the regions of interest 4000. Area 4002indicates the portion of the original inspection image 3800 that hasbeen excluded from further analysis. Additional details of the image arediscussed below.

Once all (or as many as possible) of the regions of interest have beenidentified, the ROI process may terminate and the pellet detectionprocess may begin. However, as noted above, it has been found thatcertain tubes having a conical section that transitions to ahemispherical tip may generate a sphere-cone ring 3810. In some cases,the sphere-cone ring 3810 may blend continuously with or be smaller thanthe pellet image 3806 (see, e.g., tube image 4004 in FIG. 40), in whichcase the sphere-cone ring 3810 will not be mistakenly identified as apellet image 3806. In other cases, the pellet image 3806 may be smallerthan the sphere-cone ring 3810 (see, e.g., tube image 4006 in FIG. 40).In still other cases (see, e.g., tube image 4008 in FIG. 40), the tubeimage may include only the sphere-cone ring 3810. The existence of thesphere-cone ring 3810 may make it necessary to further narrow the regionof interest to prevent the sphere-cone ring 3810 from being erroneouslyidentified as a pellet image. Any suitable method may be used to findthe sphere-cone ring 3810, and to determine whether the pellet is insidethe sphere-cone ring 3810.

An exemplary process for accounting for the sphere-cone ring 3810 is todetermine whether the inner boundary of the sphere-cone ring 3810 arevisible, and, if so, reduce the region of interest to cover only portionof the tube image 3804 that is inside the sphere-cone ring 3810. Indoing so, the process excludes the sphere-cone ring 3810 from beingconsidered a pellet. This exemplary process begins at step 3944, inwhich the ROI process searches each region of interest (as identified inthe previous steps) for the presence of an inner ring. The presence ofparallax in the image will cause the sphere-cone rings 3810 to appear atdifferent locations with respect to the center of each tube image 3804,as shown in FIGS. 38 and 40. More specifically, the sphere-cone rings3810 of the tube images 3804 that are farther from the viewing axis ofthe camera 3714 will be more offset relative from the center of the tubeimage 3804. If parallax is not accounted for, the process may mistake aportion of the sphere-cone ring 3810 for a pellet. As such, the ROIprocess accounts for this by searching at the appropriate expectedlocation of each sphere-cone ring 3810 based on the location of the tubein the array. The expected size and location of the sphere-cone rings3810 for each tube in the twenty-four tube array can be readilydetermined by testing or other means, and the location within each tubeshould remain approximately the same in each test unless there is asevere misplacement or manufacturing variance.

Referring now to FIG. 41, the ring detection process uses a radial testpattern centered at the expected center 4100 of the tube. The expectedcenter 4100 is the center is based on the known parallax relationship ofthe tube with respect to the viewing axis, and is offset from thegeometric center of the circular region of interest 4000 unless the tubeis on or nearly on the viewing axis. The radial test pattern analyzes aseries of radial traces 4102 that start a short distance 4104 from theexpected center 4100 and move in the outward radial direction. Thetraces start at a distance 4104 from the expected center 4100 because,in this embodiment, the tube images 3804 are known to include a smalldark spot 4106 caused by the gate recess of the tube (the point wherethe material passes into the injection mold during the forming process).Thus, the trace excludes the region where the gate recess spot 4106might appear. In other embodiments that lack a recess spot 4106, thisoffset may be eliminated. Any number of traces 4102 may be used. In thiscase, the radial test pattern uses thirty-six traces 4102 that arespaced at 10° angles around the expected center 4100. Also in thisembodiment, each trace starts 20 pixels from the expected center 4100(the inner dashed line in FIG. 41), and ends 90 pixels from the expectedcenter 4100 (the outer dashed line in FIG. 41). These pixel dimensionsmay, of course, be modified in other embodiments, to account for otherpixel densities and geometries.

Each trace 4102 is analyzed in the outward direction using aconventional falling edge analysis to detect a transition from ahigh-intensity region to a low-intensity region. If the entire regioninside the sphere-cone ring 3810 is filled with a pellet image 3806,then there will be no falling edges (transitions from light to dark). Ifthere is a pellet image 3806 located entirely inside the sphere-conering 3810, or no pellet image 3806 at all, then the process will detecta falling edge at the Inner edge of the sphere-cone ring 3810. The firsttime the ring detection process detects an falling edge, it records thelocation as an edge point 4108. By using only the first falling edge,the trace ignores additional falling edges that might occur as the tracecrosses objects outside the sphere-cone ring 3810, such as the image ofa water drop 4110 shown in FIG. 41. After analyzing all of the traces,the ring detection process fits a circle inside the identified edgepoints to delineate the expected location of the inner ring of thesphere-cone ring 3810.

In step 3946, the ROI process evaluates the size of the detected circleto confirm that it is within the expected size of a sphere-cone ring3810. In this example, if the detected circle has a radius of 40 pixelsto 65 pixels, then it is considered to be a valid detection of asphere-cone ring 3810. In this case, the ROI process proceeds to step3948, in which the region of interest 4000 for the tube image inquestion is cropped to the size of the circle identified by the ringdetection process. In the example shown in FIG. 41, the two top tubeimages 4000 were determined to have a qualifying sphere-cone ring 3810,and their regions of interest were cropped to the size of the circlegenerated during the ring detection process.

If no ring is detected, or if a detected ring is larger or smaller thanthe expected valid sphere-cone ring 3810 size, the ROI process moves tostep 3950 and uses the original region of interest for the tube image inquestion. In the example of FIG. 41, the two bottom tube images 4000were determined to not have a qualifying sphere-cone ring 3810, andtheir regions of interest were not cropped.

Finally, after the ROI process determines whether to use the originalregion of interest in step 3950, or the reduced region of interest instep 3948, the process continues in step 3952 to the pellet detectionprocess. The pellet detection process may use any suitable algorithm toevaluate the tube image 3804 or region of interest to determine whetherit includes a pellet image 3806 that corresponds to a sample pellet.

An example of a pellet detection process is illustrated in the flowchartof FIG. 42. The process begins in step 4200 by receiving the region ofinterest information from the ROI process. This information is used to“mask” the original image data to narrow the areas in which the pelletsare sought during the pellet detection process.

Before checking for the presence of pellets, the pellet detectionprocess scans the image to determine whether any tubes are missingaltogether. To do so, the process the process extracts the intensitydata from the original image, in step 4202, and then evaluates andreports the average intensity for each region of interest in step 4204.This process may use a simple average of the intensity value of eachpoint in each region, or other algorithms. Next, in step 4206, theprocess evaluates whether the intensity of the image in any region isgreater than would be expected if a tube was present. In thisembodiment, step 4206 determines whether any region has more than 95% ofits pixels at an intensity of 254 or greater on the 0-255 intensityscale. Any qualifying region is labeled as lacking a tube. Since thetubes in the shown embodiment are provided on tube strips 210, thatshould mean that the other three tubes on the strip are missing, andthose regions also should be identified, using the algorithm of step4206, as lacking a tube. If this is not the case, then a separate errormay be generated to indicate that the tube strip 210 is defective (e.g.,missing one tube), or that the system is not functioning properly.

After identifying any missing tubes, the process moves to step 4208 andoptionally extracts the red plane data from the original inspectionimage 3800. This may be done to change the contrast properties of theimage as described above. Alternatively, the pellet detection processmay use the full-color image, a greyscale image formed as a composite ofone or more color channels (e.g., red, green and/or blue), or otherpermutations of the original image data. As noted above, sharpening andother algorithms such as noise reduction and the like, may be performedon the image data before or during the pellet detection process.

Next, in step 4210, the process performs a threshold algorithm, such asthe conventional Niblack local thresholding algorithm, to identifydarker areas—referred to herein as “particles”—within the regions ofinterest. In step 4212, thin connections between particles are excludedto separate the particles from one another, and in step 4214 anautomatic median process is used to smooth and simplify the contours ofthe particles, and close small gaps and holes.

Starting in step 4216, various geometric criteria may be applied todetermine whether a particle is a pellet image 3806. In step 4216, abounding box 4300 is generate around each particle, such as shown inFIG. 43. The bounding box 4300 has a width W_(box), and a height H_(box)that are just large enough to enclose the particle 4302. In step 4218,the process plots the intensity of a horizontal line 4304 and a verticalline 4306 passing through the center of the bounding box 4300. Theintensity of each plot will have one value where the particle 4302 isabsent, and another value where the particle 4302 is present, as shownby segments W_(L) and H_(L). In step 4220, the process determines theratio of the Intensity portion (W_(L), H_(L)) of each line to theoverall length of each trace (equal to W_(box), H_(box)). If the valueof W_(L)/W_(box) or H_(L)/H_(box) is less than 50%, then the particle isdetermined not to be a pellet and excluded from further consideration.This step eliminates hollow particles, crescent-shaped particles, andother particles that would not have an expected pellet shape. It will beappreciated that this step, and others described herein that rely onintensity variations to delineate objects, can be performed by renderingthe particle as a high-intensity region and the background as alow-Intensity region (or simply deleting the background intensityvalue), or in the opposite manner by rendering the particles as alow-intensity region and the background as a high-intensity region—inshort, the processes and analyses described can be performed on a“negative” image or “positive” image. Thus, if the particle is renderedas a high-intensity region and the rest of the space as a low-Intensityregion, then step 4220 will operate by dividing the length of the highintensity portion of the trace by the overall width, and rejecting theparticle if the value is less than 0.5.

Next, in step 4222, the process performs a size threshold rejection byexcluding particles having an area of less than 700 pixels. Of course,in embodiments having other pixel densities and dimensional properties,different area values may be used. This step excludes the dark spot 4106caused by the gate recess, small scuffs, and other particles that aretoo small to be a likely pellet.

In step 4224, the process analyzes the ellipse ratio of each remainingparticle by dividing the length of its longest axis to the length of theaxis perpendicular to the longest axis. If the ratio of the ellipsemajor axis length to the ellipse minor axis length is greater than 9:1,then the particle is rejected from further consideration. This stepeliminates particles that are not generally round like a pellet shouldbe. This step has been found to be helpful to eliminate particlesgenerated by drops of transport media hanging on the tube walls afterdecanting in the HC2 protocol. The water drop 4110 in FIG. 41 wouldlikely be excluded in this step, if it was not excluded in step 4220 or4226 as being a hollow particle or having a large hole.

Next, in step 4226, the process eliminates particles with significantholes in them. To do so, step 4226 evaluates the bounding box image asshown in FIG. 43 and calculates the ratio of the high-intensity(particle) region to the total area (the particle region plus the holeregion), and rejects the particle from further consideration if thisvalue is less than 0.80. This step eliminates thin-walled hollowparticles.

If any of the regions of interest still includes a particle after theelimination processes described above, that particle is deemed toqualify as a genuine pellet. In step 4228, the process labels any regionof interest that passes the above criteria as having a pellet, and anyregion of interest that does not pass any one of the above criteria ashaving no pellet. FIG. 40 graphically illustrates the particles thathave passed the pellet criteria, and are deemed to be pellets 4010, incross-hatched lines. The remaining part of each tube image 4000 is notconsidered to be part of the pellet, however the particles that do notpass all of the criteria may also be represented in the image, andidentified by a different color or shading pattern. In this example, allbut two of the tubes have been found to include a pellet.

Finally, in step 4230, the process generates a data record indicatingthe status of each tube well location (and thus each correspondingsample) as lacking a tube (step 4206), having a pellet (step 4228),lacking a pellet (step 4228), or failing due to not identifying a regionof interest (step 3942).

The foregoing image processing system may be used in embodiments of aprocessing module, or as a separate stand-along processing device.However, it will be appreciated that alternative image processingequipment and methods may be used in other processing modules. Othervariations and modifications will be apparent to persons of ordinaryskill in the art in view of the present disclosure.

First Processing Example

An embodiment of a sample processing module assembled according to theembodiment of FIG. 2 was used to process human tissue samples accordingto the process described with reference to FIG. 3. Specifically, samplesin vials of PreservCyt® media were manually mixed and dispensed intoseparate tubes of a four-tube tube strip. Each tube in the strip was a 6milliliter tubes with conventional Sarstedt cone geometry. The tubestrips were manually loaded into the processing module. At this point,automated processing began. First, the processing module loaded eachtube strip into a tube strip holder that holds six tube strips. AHamilton pipettor loaded with 5 milliliter tips dispensed 400micro-liters of HC2 Sample Conversion Buffer into each tube. Next, thetube strip holder (including the tube strips and samples) was mixed on aHamilton orbital shaker operated for 30 seconds at 800 rpm on a 3millimeter orbit. The tube strip holder (and strips and samples) wasthen loaded into a BioNex centrifuge using a Hamilton iSwap transporter,and centrifuged at 2,900 gravities for 15 minutes. After centrifuging, avision system was used to confirm that a pellet was in each tube. Thetube strip holder was then conveyed to a decanting station, where eachtube strip was removed, decanted and placed back in the tube stripholder. The tube strips were decanted by rotating them in a firstdirection to a 150° angle (measured downwards from vertical), holdingthem in this position for one second, then slowly rotating them back(opposite to the first direction) to the upright starting position.Next, the tube strip holder was again placed in the vision system toconfirm that a pellet remained in each tube after decanting. The tubestrip holder was then placed on a platform and a Hamilton pipettor wasused to dispense 100 microliters of standard HC2 Specimen TransportMedium, and then 50 microliters of standard HC2 Denaturation Reagent,into each tube. Next, a cover was placed on the tube strip holder, andthe tube strip holder was placed in a Hamilton shaker and mixed for twominutes at 1,250 rpm on a 3 millimeter orbit at room temperature. Thetube strip holder was next placed on a Hamilton heater/shaker and heatedto 65° (±2°) Celsius. Fifteen minutes into heating, the heater/shakerwas operated for 30 seconds at 1,250 rpm on a 3 millimeter orbit, whileheating continued. After shaking, the tube strip holder remained on theheater/shaker and continued to incubate at 65° (±2°) for another 30minutes. At the end of the second heat cycle, the heater/shaker wasoperated at 1,250 rpm on a 3 millimeter orbit for 10 seconds, whilecontinuing to apply heat. The total incubation time, includingincubation during the mixing cycles, was 45 minutes and 40 seconds.Next, the tube strip holder was moved to a platform, and a Hamiltonpipettor was used to transfer a 75 microliter specimen from each sampletube to a respective well on a hybridization plate. Calibrators orcontrols may be processed and dispensed on the hybridization plate alongwith the samples, or processed added to the hybridization platemanually.

Second Processing Example

The process and apparatus described in the First Processing Example weremodified by making the following changes. First, the samples wereoriginally provided in SurePath™ media, rather than PreservCyt®. Second,SCB was not dispensed into the tube strip. Third, the pre-centrifugemixing step (step 310) was not performed. Fourth, the centrifuge step(step 312) was conducted at a somewhat lower gravitational load, and forapproximately ten minutes. Fifth, the samples were decanted by rotatingthe tube strip in a first direction to an angle of 210° (measureddownwards from vertical), pausing for approximately 0.5 to 1 second, andthen continuing to rotate the tube strip in the first direction untilthe tube strip was upright (i.e., a full 360° rotation with a 0.5 to 1second pause at 210°). And sixth, the incubation step (step 326) wasconducted for approximately 90 minutes, with some variation in themixing process. The remaining steps and procedures were identical tothose described in the First Processing Example.

Third Processing Example

An embodiment of a sample processing module assembled according to theembodiment of FIG. 4 was used to process human tissue samples accordingto the process described with reference to FIGS. 5 and 3. In thisexample, individual samples in PreservCyt® vials were vortexed accordingto the conventional manual HC2 procedure, and then manually loaded intovial racks. The loaded vial racks were loaded into the processingmodule, and automated processing began. The automated process began byoperating Hamilton pipettors loaded with 5 milliliter pipette tips tohydraulically mix the contents of each sample vial, and pipette 4milliliters of each mixed sample solution to a respective tube in afour-tube tube strip, such as the tube strip described in the FirstProcessing Example. From here, the processing module continuedprocessing the samples as described above in the automated processingsteps of the First Processing Example.

Fourth Processing Example

The process and apparatus described in the Third Processing Example weremodified by starting with samples in SurePath™ media and vials, ratherthan PreservCyt® media and vials. Additional changes to the process aredescribed above in the Second Processing Example. The remaining stepsand procedures were identical to those described in the Third ProcessingExample.

Unless otherwise indicated herein, the volumes and other measurementsidentified and claimed herein are intended to cover the statedmeasurement and deviations from the stated measurement that would not beexpected by persons of ordinary skill in the art to materially alter theperformance of the processes described herein, or that are generallyaccepted by the relevant persons to be an acceptable error range for themeasurement in question. Such deviations would be consideredapproximations of the stated measurement (e.g., such expected oraccepted deviations for a value of “400 microliters” would be consideredapproximately 400 microliters). Furthermore, where one value in a rangeis specifically identified as being an approximate value, it will beunderstood that the other value in the range also is an approximatevalue unless indicated otherwise.

The present disclosure describes a number of new, useful and nonobviousfeatures and/or combinations of features that may be used alone ortogether. For example, the exemplary processing modules and processingmethods may be used independently of the sample adequacy system, tubestrips, decanting systems, blotters, and heating systems describedherein, and vice versa. The multiple separate inventions stand alone andare not intended to require combination with other inventions.Furthermore, the embodiments described herein are all exemplary, and arenot intended to limit the scope of the inventions. It will beappreciated that the inventions described herein can be modified andadapted in various and equivalent ways, and all such modifications andadaptations are intended to be included in the scope of this disclosureand the appended claims.

The invention claimed is:
 1. A method for performing automated visioninspection of pelletized samples, the method comprising: inserting asample tube holder having a plurality of sample tube locations into aprocessing module, each sample tube location being configured to receivea sample tube therein; generating an inspection image of the sample tubeholder; evaluating the inspection image to determine whether a sampletube image exists for each sample tube location; evaluating theinspection image to identify a region of interest corresponding to eachsample tube location; evaluating each region of interest to determinewhether a sphere-cone ring image exists in the region of interest;generating a modified region of interest that excludes the sphere-conering if a sphere-cone ring image exists in the region of interest; andevaluating each sample tube image to determine whether a pellet islocated in each sample tube.
 2. The method of claim 1, whereingenerating the inspection image comprises illuminating a top of thesample tube holder and the inspection image comprises a silhouette imageof a bottom of the sample tube holder.
 3. The method of claim 1, whereinevaluating the image to identify a region of interest corresponding toeach sample tube location comprises identifying regions having anintensity above a predetermined threshold intensity value, and adiameter within a predetermined diameter range.
 4. The method of claim3, wherein the diameter range comprises a minimum diameter selected toexclude through-hole images corresponding to auxiliary passages throughthe sample tube holder, the auxiliary passages being separate from thesample tube locations.
 5. The method of claim 3, further comprisingidentifying a circular center of each region and comparing each circularcenter with a default grid of expected sample tube locations todetermine whether the sample tube holder is properly aligned in theinspection image.
 6. The method of claim 1, wherein evaluating the imageto identify a region of interest corresponding to each sample tubelocation comprises processing the inspection image to identify regionshaving an intensity above a threshold intensity value, an area above apredetermined minimum area, and a diameter within a predetermineddiameter range.
 7. The method of claim 1, wherein evaluating the imageto identify a region of interest corresponding to each sample tubelocation comprises: performing a first analysis to identify firstregions of interest; comparing the first regions of interest to adefault grid of expected sample tube locations to correlate a firstregion of interest with each expected sample tube location; performing asecond analysis to identify second regions of interest if the firstanalysis does not correlate a first region of interest with eachexpected sample tube location; and comparing the second regions ofinterest to the default grid of expected sample tube locations tocorrelate a second region of interest with each expected sample tubelocation not previously correlated with a first region of interest. 8.The method of claim 7, wherein the first analysis comprises identifyingfirst regions of interest having a first intensity above a firstpredetermined threshold intensity value, and a first diameter within afirst predetermined diameter range.
 9. The method of claim 8, whereinthe second analysis comprises processing the inspection image toidentify second regions of interest having a second intensity above acomputed threshold intensity value, an area above a predeterminedminimum area, and a second diameter within a second predetermineddiameter range.
 10. The method of claim 7, wherein evaluating the imageto identify a region of interest corresponding to each sample tubelocation comprises: performing a third analysis to identify thirdregions of interest if the first analysis and the second analysis do notcorrelate a first region of interest or second region of interest witheach expected sample tube location; and comparing the third regions ofinterest to the default grid of expected sample tube locations tocorrelate a third region of interest with each expected sample tubelocation not previously correlated with a first region of interest or asecond region of interest.
 11. The method of claim 10, wherein: thefirst analysis comprises identifying first regions of interest having afirst intensity above a first predetermined threshold intensity value;and the third analysis comprises identifying third regions of interesthaving a second intensity above a second predetermined thresholdintensity value; wherein the second predetermined threshold intensityvalue is less than the first predetermined threshold intensity value.12. The method of claim 1, wherein evaluating each region of interest todetermine whether a sphere-cone ring image exists in the region ofinterest comprises: performing a series of radial traces extendingradially from an expected tube center associated with each region ofinterest; searching for a respective point along each radial trace atwhich the intensity of the inspection image drops indicating thepresence of an inner edge of a sphere-cone ring; and fitting a circlewithin any identified respective points along each radial trace todelineate the expected location of the sphere-cone ring.
 13. The methodof claim 12, further comprising comparing the diameter of the circle toa predetermined range of expected sphere-cone ring diameters to validatewhether a sphere-cone ring image has been detected.
 14. The method ofclaim 12, wherein generating a modified region of interest that excludesthe sphere-cone ring comprises cropping the region of interest to thesize of the circle.
 15. The method of claim 12, wherein the expectedtube center associated with each region of interest is offset from thecenter of the region of interest according to a parallax relationshipbetween a camera generating the inspection image and an expectedlocation of a respective sample tube in the sample tube locationcorrelating to the region of interest.
 16. The method of claim 12,wherein each radial trace begins at a point offset from the expectedtube center.
 17. The method of claim 1, wherein evaluating theinspection image to determine whether a sample tube image exists foreach sample tube location comprises: evaluating the intensity profile ofa region of interest associated with each sample tube location;comparing the intensity profile with a maximum threshold intensityprofile; and identifying the respective sample tube location as lackinga sample tube if the intensity profile exceeds the maximum thresholdintensity profile.
 18. The method of claim 17, wherein the intensityprofile comprises an average intensity of the region of interest and themaximum threshold intensity profile comprises a maximum averageintensity.
 19. The method of claim 17, wherein the intensity profilecomprises an intensity value for each of a plurality of measured pointsin the region of interest and the maximum threshold intensity profilecomprises a maximum intensity value for a minimum percentage of a totalnumber of the measured points.
 20. The method of claim 1, whereinevaluating each sample tube image to determine whether a pellet islocated in each sample tube comprises: evaluating a region of interestassociated with each sample tube location to identify one or more darkregions that are darker than a threshold value; comparing the one ormore dark regions to one or more geometric criteria to determine whethereach of the one or more dark regions represents the presence of arespective pellet in each sample tube.
 21. The method of claim 20,wherein evaluating a region of interest associated with each sample tubelocation to identify one or more dark regions that are darker than athreshold value comprises: performing a local thresholding algorithm toidentify one or more dark image particles within the region of interest;excluding thin connections between the one or more dark image particles;and smoothing a contour of each of the one or more dark image particles;closing gaps and holes in each of the one or more dark image particles;and designating each or the one or more dark image particles remainingafter the foregoing steps as a respective dark region for the region ofinterest.
 22. The method of claim 20, wherein comparing the one or moredark regions to one or more geometric criteria to determine whether eachof the one or more dark regions represents the presence of a respectivepellet in each sample tube comprises: generating a respective boundingbox surrounding each of the one or more dark regions, each respectivebounding box having a box width equal to the width of the respectivedark region, and a box height equal to the height of the respective darkregion; determining the total width of the respective dark region asmeasured at 50% of the box height; determining the total height of therespective dark region as measured at 50% of the box width; anddetermining that the respective dark region does not represent thepresence of a pellet in the sample tube if the total width of therespective dark region is less than 50% of the box width or if the totalheight of the respective dark region is less than 50% of the box height.23. The method of claim 20, wherein comparing the one or more darkregions to one or more geometric criteria to determine whether each ofthe one or more dark regions represents the presence of a respectivepellet in each sample tube comprises: determining a respective area ofeach of the one or more dark regions; comparing the respective area ofeach of the one or more dark regions to a minimum threshold area; anddetermining that any of the one or more dark regions having a respectivearea that is less than the minimum threshold area does not represent thepresence of a pellet in the sample tube.
 24. The method of claim 20,wherein comparing the one or more dark regions to one or more geometriccriteria to determine whether each of the one or more dark regionsrepresents the presence of a respective pellet in each sample tubecomprises: identifying a respective ellipse ratio for each of the one ormore dark regions; comparing the respective ellipse ratio of each of theone or more dark regions to a maximum threshold ellipse ratio; anddetermining that any of the one or more dark regions having a respectiveellipse ratio that is more than the maximum threshold ellipse ratio doesnot represent the presence of a pellet in the sample tube.
 25. Themethod of claim 20, wherein comparing the one or more dark regions toone or more geometric criteria to determine whether each of the one ormore dark regions represents the presence of a respective pellet in eachsample tube comprises: generating a respective bounding box surroundingeach of the one or more dark regions, each respective bounding boxhaving a box width equal to the width of the respective dark region, anda box height equal to the height of the respective dark region;determining an area ratio of an area of the dark region to a total areaof the respective bounding box; comparing the area ratio to a minimumthreshold area ratio; and determining that any of the one or more darkregions having a respective area ratio that is less than the minimumthreshold area ratio does not represent the presence of a pellet in thesample tube.
 26. The method of claim 20, wherein comparing the one ormore dark regions to one or more geometric criteria to determine whethereach of the one or more dark regions represents the presence of arespective pellet in each sample tube comprises: eliminating any of theone or more dark regions having a shape that is dissimilar from anexpected pellet shape; eliminating any of the one or more dark regionshaving a size that is dissimilar from an expected pellet size;eliminating any of the one or more dark regions that are hollow; and ifany of the one or more dark regions remain after the foregoing steps,then indicating that a respective pellet is present in the respectivesample tube.
 27. The method of claim 26, wherein eliminating any of theone or more dark regions having a shape that is dissimilar from anexpected pellet shape comprises eliminating any of the one or more darkregions that are hollow particles, crescent-shaped, or excessivelyelongated.